Chimeric antigen receptors targeting GPC-3 and immune cells expressing same for therapeutic use

Abstract

A genetically engineered T cell that expresses a chimeric antigen receptor (CAR) targeting GPC3, and optionally has a variety of genetic edits, comprising a disrupted TRAC gene, a disrupted beta2M gene, a disrupted Regnase 1 gene, a disrupted TGFbRII gene, or a combination thereof. Also provided herein are methods of making such genetically engineered T cells and methods of using the genetically engineered T cells in the treatment of cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 356,671, filed on June 29, 2022, under 35 U.S.C. §119(e), the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on June 12, 2023, is named "095136-0766-067WO1_SEQ.xml" and is 93,246 bytes in size. Background of the Invention

[0006] Glypican-3 (GPC3) is a 580-AA heparin sulfate proteoglycan that is overexpressed in many malignancies including hepatocellular carcinoma, squamous cell lung carcinoma, ovarian cancer, and melanoma, but is absent or expressed at very low levels in normal Roswell Parkssues and normal liver tissue or cirrhosis. Capurro et al. found that 72% of HCC patients expressed GPC3, and 53% of HCC patients had significantly elevated serum levels of GPC3. The function of GPC3 has been proposed to promote the development of HCC by activating the Wnt signaling pathway. Morgan et al. found that when GPC3 on HCC cells was silenced, the proliferation and invasion ability of HCC cells were impaired, indicating that the expression of GPC3 on HCC cells is related to their proliferation and invasion. In addition, its overexpression is also a predictor of poor prognosis. Therefore, due to its strong specificity and high sensitivity, GPC3 has been used as a target for the diagnosis and treatment of HCC.

[0007] Chimeric antigen receptor (CAR) T cell therapy uses genetically modified T cells to more specifically and more effectively target and kill target cells, such as target cancer cells. After collecting T cells from the blood, these cells are transformed to contain CAR on their surface. CAR can be introduced into T cells using CRISPR / Cas9 gene editing technology. When these CAR T cells are injected into the patient, the receptor enables the T cells to kill target cells. Summary of the invention

[0008] The present disclosure is based at least in part on the development of genetically edited anti-GPC CAR-T cells that can carry one or more additional genetic 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 efficient methods for generating such genetically edited T cells by CRISPR / Cas-mediated gene editing using the guide RNA disclosed herein.

[0009] Therefore, in some aspects, the disclosure is characterized in that the genetically engineered T cell population, wherein the genetically engineered T cell comprises: (a) a nucleic acid encoding a chimeric antigen receptor (CAR) (anti-GPC3 CAR) binding to Glypican 3 (GPC3); and (b) one or more disrupted genes, comprising: (i) a disrupted T cell receptor alpha chain 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) (i)-(v) any combination of any one of the combinations. In some cases, the T cell is a human T cell. In some instances, the T cell comprises a primary human T cell.

[0010] In some embodiments, the anti-GPC3 CAR comprises: (a) an extracellular domain that binds GPC3; (b) a transmembrane domain; and (c) an intracellular domain comprising (i) a co-stimulatory signaling domain and (ii) a CD3ζ cytoplasmic signaling domain.

[0011] In some cases, the extracellular domain comprises an anti-GPC3 fragment that is an anti-GPC3 single domain antibody (e.g., VHH). Such anti-GPC3 single domain antibody may comprise a complementarity determining region (CDR) identical to that in SEQ ID NO: 9. In one example, the anti-GPC3 VHH comprises the amino acid sequence SEQ ID NO: 9.

[0012] In some cases, the extracellular domain comprises an anti-GPC3 fragment, which is an anti-GPC3 single chain variable fragment (scFv). In some examples, the extracellular domain comprises an anti-GPC3 scFv, which comprises a heavy chain variable (V H ) region and light chain variable (V L ) region, the heavy chain variable (V H) region comprises CDRs identical to those in SEQ ID NO: 22, the light chain variable (V L ) region is the same as the CDR in SEQ ID NO: 21. In a specific example, the anti-GPC3 scFv comprises a V region as shown in SEQ ID NO: 22. H and V as shown in SEQ ID NO: 21 L Such anti-GPC3 scFv may comprise the amino acid sequence of SEQ ID NO: 23. Alternatively, the anti-GPC3 scFv may comprise the amino acid sequence of SEQ ID NO: 24.

[0013] Any anti-GPC3 CAR disclosed herein may include a costimulatory domain. In some instances, the costimulatory domain is a CD28 costimulatory domain. In other instances, the costimulatory domain is a 4-1BB costimulatory domain. Alternatively or in addition, the anti-GPC3 CAR may include a transmembrane domain, which may be a CD8 transmembrane domain. In a specific example, the anti-GPC3 CAR includes an amino acid sequence of SEQ ID NO: 11, 14, 26, 29, 32 or 35. In one example, the anti-GPC3 CAR includes an amino acid sequence of SEQ ID NO: 11. In another example, the anti-GPC3 CAR includes an amino acid sequence of SEQ ID NO: 26.

[0014] In some embodiments, the genetically engineered T cells may include a disrupted TRAC gene and a disrupted β2M gene. Such genetically engineered T cells may further include a disrupted TGFBRII gene, a disrupted Reg-1 gene, a disrupted CBLB gene, or a combination thereof. For example, the genetically engineered T cells may further include (i) a disrupted TGFBRII gene and a disrupted Reg-1 gene. Alternatively, the genetically engineered T cells may further include a disrupted TGFBRII gene and a disrupted CBLB gene.

[0015] In other embodiments, the genetically engineered T cells may comprise a disrupted TGFBRII gene, a disrupted Reg-1 gene, a disrupted CBLB gene, or a combination thereof. In some examples, such genetically engineered T cells may comprise a wild-type TRAC gene, a wild-type β2M gene, or a combination thereof.

[0016] In some cases, the nucleic acid encoding the anti-GPC3 CAR can be inserted into the endogenous genetic locus of the genetically engineered T cell. For example, the endogenous genetic locus is located in the destroyed TRAC gene, the destroyed β2M gene, the destroyed TGFbRII gene, the destroyed Reg-1 gene, or the destroyed CBLB gene. In some examples, the endogenous genetic locus is located in the destroyed TRAC gene. In a specific example, the destroyed TRAC gene includes a deletion of SEQ ID NO: 58, which can be replaced by a nucleotide sequence encoding an anti-GPC3 CAR.

[0017] In other aspects, provided herein is a method for producing a population of genetically engineered T cells, the method comprising: (a) delivering to a population of T cells (i) one or more RNA-guided nucleases, (ii) one or more guide RNAs that target a T cell receptor alpha chain constant region (TRAC) gene (TRAC gRNA), a beta-2 microglobulin (β2M) gene (β2M gRNA), a TGFbRII gene (TGFBRII gRNA), a Regnase-1 (Reg1) gene (Reg1 gRNA), and / or a Casitas B lineage lymphoma proto-oncogene-B (CBLB) gene (CBLB gRNA); and (iii) a vector comprising a nucleic acid encoding an anti-GPC3 CAR, such as those disclosed herein; and (b) producing a population of engineered T cells that express an anti-GPC3 CAR and comprise one or more disrupted TRAC genes, β2M genes, TGFBRII genes, Reg1 genes, and CBLB genes.

[0018] In some instances, the T cell population comprises human T cells, such as human primary T cells. In some instances, the T cell population is obtained from one or more healthy human donors. Alternatively, the T cell population is obtained from a human patient with a GPC3+ cancer.

[0019] In some embodiments, step (a) includes delivering the TRAC gRNA and the β2MgRNA to the T cell group. In some cases, step (a) further includes delivering TGFBRII gRNA, Reg1 guide, CBLB guide or a combination thereof to the T cell group. For example, step (a) may further include delivering (i) TGFBRII gRNA and Reg1 guide to the T cell group. Alternatively, step (a) may include delivering TGFBRII guide and CBLB guide to the T cell group. In other cases, step (a) includes (e.g., consisting of) delivering TGFBRII gRNA, Reg1 guide, CBLB guide or a combination thereof to the T cell group.

[0020] In some instances, 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. In some cases, the TRAC guide may comprise a scaffold sequence (e.g., those disclosed herein). In some cases, the TRAC guide may comprise one or more modifications. An exemplary TRAC guide may comprise the nucleotide sequence of SEQ ID NO:37 or SEQ ID NO:38.

[0021] In some instances, the β2M guide is specific for a β2M gene target sequence comprising a nucleotide sequence of SEQ ID NO: 60. Such a β2M guide may comprise a spacer region comprising a nucleotide sequence of SEQ ID NO: 43. In some cases, the β2M guide may comprise a scaffold sequence (e.g., those disclosed herein). In some cases, the β2M guide may comprise one or more modifications. An exemplary β2M guide may comprise a nucleotide sequence of SEQ ID NO: 41 or SEQ ID NO: 42.

[0022] In some examples, the TGFBRII guide is specific for a TGFBRII gene target sequence comprising the nucleotide sequence of SEQ ID NO: 62. Such a TGFBRII guide can comprise a spacer region comprising the nucleotide sequence of SEQ ID NO: 47. In some cases, the TGFBRII guide can comprise a scaffold sequence (e.g., those disclosed herein). In some cases, the TGFBRII guide can comprise one or more modifications. An exemplary TGFBRII guide can comprise the nucleotide sequence of SEQ ID NO: 45 or SEQ ID NO: 46.

[0023] In some instances, the Reg1 guide is specific for a Reg1 gene target sequence comprising the nucleotide sequence of SEQ ID NO: 64. Such a Reg1 guide may comprise a spacer region comprising the nucleotide sequence of SEQ ID NO: 51. In some cases, the Reg1 guide may comprise a scaffold sequence (e.g., those disclosed herein). In some cases, the Reg1 guide may comprise one or more modifications. An exemplary Reg1 guide may comprise the nucleotide sequence of SEQ ID NO: 49 or SEQ ID NO: 50.

[0024] In some instances, the CBLB guide is specific for a CBLB gene target sequence comprising the nucleotide sequence of SEQ ID NO: 66. Such a CBLB guide may comprise a spacer region comprising the nucleotide sequence of SEQ ID NO: 55. In some cases, the CBLB guide may comprise a scaffold sequence (e.g., those disclosed herein). In some cases, the CBLB guide may comprise one or more modifications. An exemplary CBLB guide may comprise the nucleotide sequence of SEQ ID NO: 53 or SEQ ID NO: 54.

[0025] In any of the methods disclosed herein, the one or more RNA-guided nucleases may comprise a Cas9 nuclease. In some examples, the Cas9 nuclease is a Streptococcus pyogenes Cas9 nuclease.

[0026] Alternatively or additionally, the vector used in (a)(iii) of any method disclosed herein may include a donor template, wherein the flank of the nucleic acid encoding the anti-GPC3 CAR is an upstream fragment and a downstream fragment, and wherein the upstream fragment and the downstream fragment are homologous to the endogenous genetic locus of the T cell, allowing the nucleic acid encoding the anti-GPC3 CAR to be inserted into the endogenous genetic locus. Exemplary endogenous genetic 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 instances, the endogenous genetic locus is located within the disrupted TRAC gene. In this case, the upstream fragment may be SEQ ID NO: 71, and / or the downstream fragment may be SEQ ID NO: 74.

[0027] In some examples, the vector in (a)(iii) is a viral vector, such as an adeno-associated virus (AAV) vector or a lentiviral vector.

[0028] In other aspects, provided herein are methods of treating cancer in a subject, comprising administering to a subject in need thereof any of the genetically engineered T cell populations disclosed herein. In some embodiments, the subject is a human patient with 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.

[0029] In some instances, the genetically engineered T cell population is allogeneic to the human patient. Such genetically engineered T cell populations may 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, the genetically engineered T cell population is autologous to the human patient. Such genetically engineered T cell populations may have a disrupted TGFBRII gene, a disrupted Reg-1 gene, and / or a disrupted cbl-b gene, and optionally a wild-type TRAC and / or β2M gene.

[0030] Further, the present disclosure provides a chimeric antigen receptor (anti-GPC3 CAR) that binds Glypican 3 (GPC3) as disclosed herein. In some cases, the anti-GPC3 CAR may further comprise an N-terminal signal peptide. Exemplary anti-GPC3 CAR polypeptides (with or without signal peptides) are provided in Table 1, all of which are within the scope of the present disclosure. Also provided herein are nucleic acids encoding anti-GPC3 CARs and host cells comprising such nucleic acids. In some cases, the nucleic acid is located in a suitable vector, such as a viral vector such as an AAV vector or a lentiviral vector.

[0031] In addition, any genetically engineered T cells expressing anti-GPC3 CAR are provided herein for inhibiting GPC3+ disease cells such as cancer cells and for treating diseases associated with such disease cells, such as various cancers disclosed herein. It is also within the scope of the present disclosure that genetically engineered T cells expressing anti-GPC3 CAR are used to prepare drugs for intended therapeutic applications.

[0032] The details of one or more embodiments of the invention are set forth in the following description. Other features and advantages of the invention will become apparent from the following drawings and detailed description of several embodiments and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1A-1B Contains graphs showing evaluation of anti-GPC3 CAR expression and TRAC and B2M (beta 2 microglobulin) gene editing efficiency. Figure 1A : Graphs showing expression of anti-GPC3 CAR in T cells transduced with the indicated CAR constructs. Figure 1B : Graph showing the efficiency of TRAC and B2M gene KO in T cells expressing the indicated CAR constructs.

[0034] Figure 2A-2B Contains graphs showing the frequency of CD4+ and CD8+ T cells, as well as graphs assessing the relative enrichment of T cell populations based on CCR7 / CD45RA staining. Figure 2A: Graph showing the frequency of CD4+ and CD8+ T cells in cohorts of cells expressing the indicated CAR constructs. Figure 2B : Graph showing the relative enrichment of Teff, Tcm, Tnaive, and TEMRA in a cell cohort.

[0035] Figure 3 is a graph showing the expression of immune checkpoint molecules programmed cell death protein 1 (PD1) and lymphocyte activation gene 3 (Lag3) in T cells expressing the indicated CAR constructs.

[0036] Figures 4A-4C Contains graphs showing the cytotoxic effects of T cells expressing anti-GPC3 CAR. Figure 4A : Shows the effect of anti-GPC3 CAR T cells on GPC3 at the indicated T cell / target cell ratios. Hi Figure 3. Cytotoxicity of HepG2 target cells. Figure 4B : Shows the effect of anti-GPC3 CAR T cells on GPC3 at the indicated T cell / target cell ratios. Lo Figure 3. Cytotoxicity of Huh7 target cells. Figure 4C : Shows the effect of anti-GPC3 CAR T cells on GPC3 at the indicated T cell / target cell ratios. - Figure 3. Cytotoxicity of A498 target cells.

[0037] Figure 5 It is shown that GPC3 + Graph of the effect of anti-GPC3 CAR T cell administration on tumor volume in NSG mice bearing HepG2 cancer cells.

[0038] Figure 6 is a graph showing in vitro expansion of anti-GPC3 CAR T cells after electroporation.

[0039] Figure 7 TRAC with additional disruption of TGFbRII / Reg-1 (T+R) or TGFbRII / cbl-b (T+C) was evaluated. - / B2M - / Diagram of anti-GPC3 CAR expression in anti-GPC3 CAR T cells.

[0040] Figures 8A-8C Included display Figure 7 Figure 2. Graph of the editing efficiency of TRAC, B2M, TGFbRII, and Regnase-1 (Reg-1) genes in CAR T cells described in Figure 2. Fig. 8A : Graph showing the TRAC and B2M gene KO efficiency in CAR T cells. Figure 8B: A graph showing the efficiency of TGFbRII gene KO in TGFbRII / Reg-1(T+R)CAR T cells. Figure 8C : A graph showing the KO efficiency of Reg-1 gene in TGFbRII / Reg-1 (T+R)CAR T cells.

[0041] Fig. 9 It is displayed in Figure 7 Graph of the efficiency of cbl-b KO in TGFbRII / cbl-b KO (T+C) CAR T cells analyzed by western blot as described in .

[0042] Fig.10 It is displayed in Figure 7 Graph of the frequencies of CD4+ and CD8+ T cells in the CAR T cells described in.

[0043] Figures 11A-11B Included in the display Figure 7 Figure 2 shows a graph of the cytotoxic effect of CAR T cells described in Figure 2. Fig.11A : Shows the effect of anti-GPC3 CAR T cells on GPC3 at the indicated T cell / target cell ratios. Hi Figure 3. Cytotoxicity of HepG2 target cells. Fig. 11B : Graph showing the cytotoxicity of anti-GPC3 CAR T cells against GPC3-A498 target cells at the indicated T cell / target cell ratios.

[0044] Figures 12A to 12B Includes display evaluation Fig.11A Described in GPC3 Hi Figure 3. Cytokine secretion of CAR T cells incubated with HepG2 target cells. Fig. 12A : Graphs showing IFN-γ secretion. Fig. 12B : Diagram showing granzyme A secretion. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present disclosure aims to establish genetically engineered anti-GPC3 CAR-T cells with improved characteristics, such as growth activity, persistence, reduced T cell exhaustion and / or enhanced efficacy. The anti-GPC3 CAR-T cells disclosed herein may include a variety of genetic editing of endogenous genes, such as destruction of the TRAC gene, β2M gene, TGFBRII gene, Reg1 gene and / or cbl-b gene, to prepare cells suitable for allogeneic immune cell therapy or autologous immune cell therapy, and to achieve characteristics that can improve the therapeutic effect. For example, β2M destruction can reduce the risk of host-versus-graft reaction or prevent host-versus-graft reaction, while TRAC destruction can reduce the risk of graft-versus-host reaction or prevent graft-versus-host reaction. Therefore, the anti-GPC3 CAR-T cells disclosed herein with a destroyed TRAC gene and / or a destroyed β2M gene can be suitable for allogeneic cell therapy. Alternatively or in addition, TGFBRII disruption may reduce the immunosuppressive effects of transforming growth factor β (TGF-β) in the tumor microenvironment, while Reg1 disruption may improve CAR-T cell function by long-term persistence of powerful effector functions. Further, CAR-T cells with disrupted cblb genes as disclosed herein also exhibit enhanced anti-tumor activity and prolonged survival. In some cases, anti-GPC3 CAR-T cells with disrupted TGFBRII genes, Reg1 genes and / or cbl-b genes and wild-type TRAC and β2M genes may be suitable for autologous cell therapy.

[0047] As provided herein, the anti-GPC3 CAR-T cells disclosed herein show high editing efficiency for target genes such as TRAC, β2M, Reg-1, TGFBRII and / or cbl-b, and there is no significant interference with the expression of immune checkpoint molecules such as PD1 and LAG-3. Further, anti-GPC3 CAR-T cells show high cytotoxicity for GPC3+ cells and GPC3+ tumors both in vitro and in vivo. Additional gene editing, such as the destruction of Reg-1, TGFBRII and / or cbl-b genes, shows no effect on CAR-T cell characteristics (including cell growth and CD4:CD8 cell ratio). CAR-T cells with additional gene editing show good proliferation and specific cytotoxicity to the target, and are considered to be phenotypically normal. In addition, as observed in animal models, such CAR-T cells show enhanced anti-tumor activity. Therefore, the anti-GPC3 CAR-T cells disclosed herein, such as those with one or more gene edits (disruption of Reg-1, TGFBRII and / or cbl-b genes), are expected to achieve excellent anti-tumor efficacy in autologous cell therapy or allogeneic cell therapy.

[0048] Such T cells can use real T cells as starting materials, such as untransformed T cells, terminally differentiated T cells, T cells with stable genomes, and / or T cells that rely on cytokines and growth factors for proliferation and amplification. Alternatively, such T cells can use T cells generated by precursor cells, such as hematopoietic stem cells (e.g., iPSCs), for example, in vitro culture. T cells disclosed herein can confer one or more benefits to both CAR-T cell manufacturing and clinical applications.

[0049] Other advantageous features associated with disruption of the Reg1 gene and / or TGFBRII gene can be found in WO / 2022 / 064428, the relevant disclosure of which is incorporated herein by reference for the purposes and subject matter cited herein.

[0050] Thus, provided herein are anti-GPC3 CAR-T cells that may have improved persistence and enhanced anti-tumor activity, methods of generating such T cells, and therapeutic applications of such T cells in eliminating GPC3+ disease cells such as cancer cells (e.g., GPC3+ hepatocellular carcinoma cells).

[0051] I. Anti-GPC3 CAR-T cells with enhanced characteristics

[0052] Glypican-3 (GPC3) is known to be an important cell surface marker for many malignant tumors, including hepatocellular carcinoma, squamous cell lung carcinoma, ovarian cancer and melanoma. GPC3 has been used as a target for the diagnosis and treatment of such GPC3+ tumors such as liver cancer, gastric cancer, colorectal cancer, lung cancer, ovarian cancer, skin cancer, thyroid cancer, etc. due to its strong specificity and high sensitivity.

[0053] In some aspects, anti-GPC3 CAR-T cells are provided herein, which may have a variety of genetic modifications to improve CAR-T cell function and thus improve the therapeutic efficacy of allogeneic cell therapy or autologous cell therapy. The genetically engineered T cells provided herein express a chimeric antigen receptor (CAR) binding to GPC3 and optionally have a variety of genetic editing for endogenous genes such as TRAC gene, β2M gene, Reg1 gene, TGFBRII gene or a combination thereof. In some cases, the anti-GPC3 CAR-T cells disclosed herein include destroyed TRAC and β2M genes, as well as Reg1, TGFBRII and / or cbl-b genes. In other cases, the anti-GPC3 CAR-T cells disclosed herein include destroyed Reg1, TGFBRII and / or cbl-b genes and may have wild-type TRAC and β2M genes.

[0054] Genetically engineered T cells can be derived from a suitable source, for example, parental T cells (e.g., unedited wild-type T cells) obtained from one or more mammalian donors. In some instances, parental T cells are primary T cells (e.g., untransformed and terminally differentiated T cells) obtained from one or more human donors. Alternatively, parental T cells may be differentiated from precursor T cells obtained from one or more suitable donors or stem cells (such as hematopoietic stem cells or induced pluripotent stem cells iPSC), which can be cultured in vitro.

[0055] Any genetically engineered T cell can be generated by gene editing (including genome editing), which is a genetic engineering in which nucleotides / nucleic acids are inserted, deleted and / or replaced in a DNA sequence, such as in the genome of a target cell. Targeted gene editing can be inserted, deleted and / or replaced at a preselected site in the target cell genome (for example, in a target gene or target DNA sequence). When the sequence of an endogenous gene is edited, for example, by deletion, insertion or replacement of nucleotides / nucleic acids, an endogenous gene containing the affected sequence may be knocked out due to sequence changes. Therefore, targeted editing can be used to destroy endogenous gene expression. "Targeted integration" refers to a process involving inserting one or more exogenous sequences, deleting or not deleting endogenous sequences at an insertion site. When there is a donor template containing an exogenous sequence, targeted integration can be produced by targeted gene editing.

[0056] A. Genes edited

[0057] In some aspects, the present disclosure provides genetically engineered T cells, which may comprise a disrupted Reg1 gene, a disrupted TGFBRII gene, a disrupted cbl-b gene, a disrupted TRAC gene, and / or a disrupted β2M gene.

[0058] As used herein, "disrupted gene" refers to a gene that contains an insertion, deletion or substitution relative to an endogenous gene, so that the expression of a functional protein from an endogenous gene is reduced or inhibited. As used herein, "disrupting a gene" refers to a method of inserting, deleting or replacing at least one nucleotide / nucleic acid in an endogenous gene, so that the expression of a functional protein from an endogenous gene is reduced or inhibited. Methods for disrupting genes are known to those skilled in the art and are described herein.

[0059] In some embodiments, cells comprising a disrupted gene do not express (e.g., on the cell surface) detectable levels (e.g., in an immunoassay using an antibody that binds to the encoded protein or by flow cytometry) of a protein encoded by the disrupted gene. Cells that do not express detectable levels of a protein can be referred to as knockout cells.

[0060] Reg1 gene editing

[0061] In some embodiments, the genetically engineered T cells may comprise a gene involved in the destruction of mRNA decay. Such a gene may be Reg1. Reg1 contains a zinc finger motif, binds RNA and exhibits ribonuclease activity. Reg1 plays a role in both immune and non-immune cells, and its expression can be rapidly induced under a variety of conditions, including microbial infection, inflammatory cytokine treatment, and chemical or mechanical stimulation. The human Reg1 gene is located on chromosome 1p34.3. Additional information can be found under GenBank's Gene ID: 80149.

[0062] In some instances, genetically engineered T cells may include a destroyed Reg1 gene so that the expression of Reg1 in T cells is substantially reduced or completely eliminated. The destroyed Reg1 gene may include one or more genetic editors that destroy the expression of the Reg1 gene at one or more suitable target sites (e.g., in a coding region or in a non-coding regulatory region such as a promoter region). Such target sites can be identified based on gene editing methods for the preparation of genetically engineered T cells. Exemplary target sites for genetic editing may include exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or a combination thereof. In some instances, one or more genetic editors may occur in exon 2 or exon 4. Such genetic editing can be induced by using CRISPR / Cas technology using suitable guide RNAs (e.g., those listed in Table 2). See also WO / 2022 / 064428, for purposes and themes cited herein, its related disclosures are incorporated herein by reference.

[0063] Disruption of the Reg1 gene improved T cell function by enhancing long-term persistence and maintaining robust effector function.

[0064] TGFBRII gene editing

[0065] In some embodiments, the genetically engineered T cells may comprise a disrupted TGFBRII gene, which encodes a type II transforming growth factor receptor (TGFBRII). The TGFBRII receptor is a family of serine / threonine kinase receptors that participate in the TGF signaling pathway. These receptors bind to growth factors and cytokine signaling proteins in the TGF family, such as TGFβ (TGFβ1, TGFβ2, and TGFβ3), bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), activins and inhibins, myostatin, anti-Mullerian hormone (AMH), and NODAL.

[0066] In some examples, genetically engineered T cells may include a destroyed TGFBRII gene so that the expression of TGFBRII in T cells is substantially reduced or completely eliminated. The destroyed TGFBRII gene may include one or more genetic editing at one or more suitable target sites (e.g., in a coding region or a non-coding regulatory region such as a promoter region), which destroys the expression of the TGFBRII gene. Such target sites can be identified based on gene editing methods for the preparation of genetically engineered T cells. Exemplary target sites for genetic editing may include exon 1, exon 2, exon 3, exon 4, exon 5, or a combination thereof. In some examples, one or more genetic editing may occur in exon 4 and / or exon 5. Such genetic editing may be induced by using a gene editing technique (e.g., CRISPR / Cas technology) using a suitable guide RNA (e.g., those listed in Table 2). See also WO / 2022 / 064428, for purposes and themes cited herein, its related disclosures are incorporated herein by reference.

[0067] Disruption of the TGFBRII gene can eliminate the surface expression of TGFBRII in the tumor microenvironment and reduce the immunosuppressive effects of transforming growth factor β (TFG-β).

[0068] cbl-b gene editing

[0069] In some embodiments, the genetically engineered T cells may include a disrupted Cbl proto-oncogene B (cbl-b) gene. The CBLB protein contains a zinc finger motif, binds RNA and exhibits ribonuclease activity. CBLB plays a role in both immune and non-immune cells, and can be rapidly induced to express under a variety of conditions, including microbial infection, inflammatory cytokine therapy, and chemical or mechanical stimulation. The human cbl-b gene is located on chromosome GRCh38.p13. Additional information can be found under GenBank's Gene ID: 868.

[0070] In some instances, genetically engineered T cells may include the cbl-b gene of destruction, so that the expression of cbl-b in T cells is substantially reduced or completely eliminated. The cbl-b gene of destruction may include one or more genetic editors at one or more suitable target sites (e.g., in coding regions or non-coding regulatory regions such as promoter regions), which destroy the expression of cbl-b genes. Such target sites can be identified based on gene editing methods, for the preparation of genetically engineered T cells. Exemplary target sites for genetic editing may include exon 2, exon 7, exon 9, exon 11, exon 12, or a combination thereof. In some instances, one or more genetic editors may occur in exon 2. In other instances, one or more genetic editors may occur in exon 7. In yet other instances, one or more genetic editors may occur in exon 9. Such genetic editing may be induced by using the CRISPR / Cas technology of suitable guide RNA (e.g., those listed in Table 2). See also U.S. Provisional Application No. 63 / 292,715, the relevant disclosure of which is incorporated herein by reference for the purposes and subject matter cited herein.

[0071] β2M gene editing

[0072] In some embodiments, the genetically engineered T cells disclosed herein may further include a disrupted β2M gene. β2M is a common (unchanged) component of the MHC I complex. Destroying its expression by gene editing will prevent the host from reacting with therapeutic allogeneic T cells, thereby resulting in increased persistence of allogeneic T cells. In some embodiments, the expression of the endogenous β2M gene is eliminated to prevent host-versus-graft reactions.

[0073] In some embodiments, the edited β2M gene may comprise a nucleotide sequence selected from the following sequences in Table 2. It is known to those skilled in the art that different nucleotide sequences in edited genes (such as edited β2M genes) can be generated by a single gRNA (such as the gRNA (β2M-1) listed in Table 2). See also WO2019097305, the relevant disclosure of which is incorporated herein by reference for the purposes and subject matter cited herein.

[0074] TRAC gene editing

[0075] In some embodiments, the genetically engineered T cells disclosed herein may further include a destroyed TRAC gene. This destruction causes TCR function loss, and makes the engineered T cells non-alloreactive and suitable for allogeneic transplantation, thereby minimizing the risk of graft-versus-host disease. In some embodiments, the expression of endogenous TRAC genes is eliminated to prevent graft-versus-host reactions. See also WO2019097305, for the purposes and themes cited herein, its relevant disclosures are incorporated herein by reference.

[0076] Genetic editing of such TRAC genes can be induced by gene editing techniques (e.g., CRISPR / Cas technology) using appropriate guide RNAs (e.g., those listed in Table 2).

[0077] It should be understood that more than one suitable target site / gRNA can be used for each target gene disclosed herein, such as those known in the art or disclosed herein. Other examples can be found in, for example, WO2019097305, and its relevant disclosure is incorporated herein by reference for the purposes and themes cited herein.

[0078] In some cases, the nucleic acid encoding the anti-GPC3 CAR can be inserted into the TRAC gene to disrupt the expression of the TRAC gene. For example, the nucleic acid encoding the CAR can replace the target site of the gRNA for gene editing by CRISPR / Cas9 (e.g., replacing the fragment containing SEQ ID NO:58 in the TRAC gene).

[0079] B. Anti-GPC3 Chimeric Antigen Receptor (CAR)

[0080] Chimeric antigen receptor (CAR) refers to an artificial immune cell receptor that is engineered to recognize and bind to antigens expressed by undesirable cells (e.g., disease cells, such as cancer cells). T cells expressing CAR polypeptides are referred to as CAR T cells. CAR has the ability to redirect T cells specifically and reactively to selected targets in a non-MHC restricted manner. Non-MHC restricted antigen recognition gives CAR-T cells the ability to recognize antigens independently of antigen processing, thereby bypassing the main mechanism of tumor escape. In addition, when expressed on T cells, CAR advantageously does not dimerize with endogenous T cell receptor (TCR) α and β chains.

[0081] There are multiple generations of CAR, each of which contains different components. The first generation of CAR connects the antibody-derived scFv to the intracellular signaling domain of CD3ζ (ζ or z) of the T cell receptor through a hinge and a transmembrane domain. The second generation of CAR includes additional costimulatory domains, such as CD28, 4-1BB (41BB) or ICOS, to provide costimulatory signals. The third generation of CAR contains two costimulatory domains fused to the TCR CD3ζ chain (e.g., a combination of CD27, CD28, 4-1BB, ICOS or OX40). Maude et al., Blood. 2015; 125 (26): 4017-4023; Kakarla and Gottschalk, Cancer J. 2014; 20 (2): 151-155). Any different generation of CAR constructs are within the scope of the present disclosure.

[0082] In general, CAR is a fusion polypeptide comprising an extracellular domain that recognizes a target antigen (e.g., a single-chain fragment (scFv) of an antibody or other antibody fragment) and an intracellular domain that comprises a signaling domain (e.g., CD3ζ) of a T cell receptor (TCR) complex, and a co-stimulatory domain in most cases. (Enblad et al., Human Gene Therapy. 2015; 26(8): 498-505). The CAR construct may further include a hinge and a transmembrane domain between the extracellular domain and the intracellular domain, and a signal peptide for surface expression at the N-terminus. Table 1 provides exemplary signal peptides. Other signal peptides may be used.

[0083] (i) Antigen binding extracellular domain

[0084] The antigen binding extracellular domain is the region of the CAR polypeptide exposed to the extracellular fluid when CAR is expressed on the cell surface. In some cases, the signal peptide can be located at the N-terminus to promote cell surface expression. In some cases, the extracellular antigen binding domain can be an antibody fragment binding GPC3 (e.g., human GPC3), for example, a single-chain variable fragment (scFv) or a single-domain antibody fragment such as an antibody fragment (VHH) of only a heavy chain.

[0085] Anti-GPC3 scFv fragment

[0086] In some embodiments, the antigen binding domain may be a single chain variable fragment (scFv, which may comprise an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L )(in either direction). In some cases, V H and V LThe fragments can be connected by peptide linkers. In some embodiments, the linker comprises hydrophilic residues with glycine and serine stretches for flexibility and glutamic acid and lysine stretches for increased solubility. The scFv fragment retains the antigen binding specificity of the parent antibody from which the scFv fragment is derived. In some embodiments, the scFv can comprise a humanized V H and / or V L In other embodiments, the V domain of the scFv H and / or V L The domain is entirely human.

[0087] In some embodiments, the antigen-binding extracellular domain can be a single-chain variable fragment (scFv) that binds to the GPC3 antigen, as disclosed herein. The scFv can comprise an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ), which may optionally be connected via a flexible peptide linker. In some cases, a scFv may have a V H To V L direction (from N-terminus to C-terminus). Alternatively, the scFv may have a V L To V H Direction (from N-terminus to C-terminus).

[0088] In some examples, the antigen-binding extracellular domain can be a single chain variable fragment (scFv) that binds to human GPC3. In some cases, the anti-GPC3 scFv can comprise (i) a heavy chain variable region (V H ), which comprises a heavy chain complementarity determining region (CDR) identical to the heavy chain CDR in SEQ ID NO: 22; and (ii) a light chain variable region (V L ), which comprises a light chain CDR identical to the light chain CDR in SEQ ID NO: 21. See Table 1 below. In some specific examples, the anti-GPC3 antibodies disclosed herein may comprise a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 as shown in SEQ ID NO: 18-20, respectively, as determined by the Kabat method. Alternatively or in addition, the anti-GPC3 antibodies disclosed herein may comprise a light chain CDR1, a light chain CDR2, and a light chain CDR3 as shown in SEQ ID NO: 15-17, as determined by the Kabat method. In a specific example, the anti-GPC3 scFv may include a V comprising the amino acid sequence of SEQ ID NO: 22. H and V comprising the amino acid sequence of SEQ ID NO: 21 L . See Table 1 below.

[0089] With the same V H and / or V LCDRs of two antibodies mean that their CDRs are identical when determined by the same method (eg, the Kabat method, Chothia method, AbM method, contact method, or IMGT method known in the art). See, e.g., Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., 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 / .

[0090] In other embodiments, the anti-GPC3 scFv may be a functional variant derived from the exemplary anti-GPC3 antibodies listed in Table 1. Such functional variants are substantially similar to the exemplary anti-GPC3 antibodies in both structure and function. The functional variant comprises a V substantially the same as Ab-1 or Ab-2. H and V L For example, relative to an exemplary anti-GPC3 antibody, it may comprise only up to 8 (e.g., 8, 7, 6, 5, 4, 3, 2, or 1) amino acid residue changes in the total CDR region and bind with substantially similar affinity (e.g., with a K of the same order). D In some cases, the functional variant may have the same heavy chain CDR3 as the exemplary anti-GPC3 antibody, and optionally the same light chain CDR3 as the exemplary anti-GPC3 antibody. Such anti-GPC3 scFv may comprise a V variant having CDR amino acid residue changes (e.g., up to 5, e.g., 5, 4, 3, 2, and 1) only in the heavy chain CDR1 and / or CDR2 as compared to the exemplary anti-GPC3 antibody. H Alternatively or in addition, the anti-scFv antibody may further comprise a V fragment having CDR amino acid residue changes (e.g., up to 5, e.g., 5, 4, 3, 2, and 1) only in the light chain CDR1 and / or CDR2 as compared to the exemplary anti-GPC3 antibody. L In some examples, the amino acid residue changes can be conservative amino acid residue substitutions.

[0091] In some examples, any change in one or more CDR regions may be a conservative substitution. 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 known to those of ordinary skill in the art for changing polypeptide sequences, such as those found in references compiling such methods, such as Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, edited by FM Ausubel et al., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions between 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.

[0092] In some embodiments, the anti-GPC3 scFv derived from the exemplary anti-GPC3 antibody can be from N-terminus to C-terminus, V H -Connector-V L In some examples, the anti-GPC3 scFv comprises V of SEQ ID NO: 22. H Fragment and V of SEQ ID NO:21 L In a specific example, the anti-GPC3 scFv in any anti-GPC3 CAR may comprise the amino acid sequence of SEQ ID NO: 23. Alternatively, the anti-GPC3 scFv derived from the exemplary anti-GPC3 antibody may be from N-terminus to C-terminus, V L -Connector-V H In some examples, the anti-GCP3 scFv in any anti-GPC3 CAR may comprise the amino acid sequence of SEQ ID NO: 24. In some cases, the anti-GPC3 scFv may have at least 85% (e.g., at least 90%, at least 95% or more) sequence identity with SEQ ID NO: 23 or SEQ ID NO: 24.

[0093] 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 from Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. This algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed using the XBLAST program with score = 50 and word length = 3 to obtain amino acid sequences homologous to the protein molecule of interest. When there is a gap between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0094] Anti-GPC3 single domain antibody fragment

[0095] In some embodiments, the antigen binding domain can be a single domain antibody fragment. Single domain antibodies, also known as nanobodies, are small antigen binding fragments containing only one heavy chain or light chain variable region (in contrast to conventional antibodies having both heavy and light chain variable regions). In some cases, the single domain antibodies provided herein are heavy chain-only antibodies (VHH antibodies) containing a single heavy chain variable region.

[0096] Like conventional antibodies, single domain antibodies such as VHH antibodies contain hypervariable regions, also called "complementarity determining regions" ("CDRs"), interspersed with more conserved regions, called "framework regions" ("FRs"). VHH antibodies are usually composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs can be precisely identified using methods 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 well known in the art. See, e.g., Kabat, EA 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).

[0097] In some embodiments, the anti-GPC3 antibody portions disclosed herein may have the same complementary determining regions (CDRs) as the exemplary anti-GPC3 VHH antibodies provided in Table 1 herein. Such anti-GPC3 VHH antibodies may have CDR1, CDR2, and CDR3 (following the Kabat definition) comprising SEQ ID NOs: 6-8, respectively. In some embodiments, the anti-GPC3 antibody portions disclosed herein may have a certain level of sequence identity (e.g., at least 80%, such as at least 85%, at least 90%, at least 95% or more) 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 changes (e.g., up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid changes) relative to the exemplary anti-GPC3 VHH antibodies. The amino acid residue changes disclosed in the present disclosure (e.g., in the framework regions and / or in the CDRs) may be conservative amino acid residue substitutions. In one example, the anti-GPC3 antibody portion may comprise the amino acid sequence of SEQ ID NO: 9.

[0098] (ii) Transmembrane domain

[0099] The anti-GPC3 CAR polypeptide disclosed herein may contain a transmembrane domain, which may be a hydrophobic alpha helix that spans the membrane. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. The transmembrane domain can provide stability for an anti-GPC3 CAR containing it.

[0100] In some embodiments, the transmembrane domain of the anti-GPC3 CAR provided herein can be a CD8 transmembrane domain. In other embodiments, the transmembrane domain can be a CD28 transmembrane domain. In yet other embodiments, the transmembrane domain is a chimera of CD8 and CD28 transmembrane domains. Other transmembrane domains provided herein can be used. In some embodiments, the transmembrane domain is a CD8a extracellular + transmembrane domain containing a sequence of SEQ ID NO: 2, as shown in Table 1 below. Other transmembrane domains can be used.

[0101] (iii) Hinge domain

[0102] In some embodiments, the hinge domain may be located between the extracellular domain (comprising 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 may be any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain and / or the cytoplasmic domain in the polypeptide chain. The hinge domain may act to provide flexibility to the anti-GPC3 CAR or its domain, or to prevent steric hindrance of the anti-GPC3 CAR or its domain.

[0103] In some embodiments, the hinge domain may comprise up to 300 amino acids (e.g., 10 to 100 amino acids, or 5 to 20 amino acids). In some embodiments, one or more hinge domains may be included in other regions of the anti-GPC3 CAR. In some embodiments, the hinge domain may be a CD8 hinge domain. Other hinge domains may be used.

[0104] (iv) Intracellular signaling domain

[0105] Any anti-GPC3 CAR construct contains one or more intracellular signaling domains (e.g., CD3ζ, and optionally one or more co-stimulatory domains), which are the functional ends of the receptors. After antigen recognition, the receptors aggregate and transmit signals to the cells. CD3ζ is the cytoplasmic signaling domain of the T cell receptor complex.

[0106] CD3ζ contains three (3) immunoreceptor tyrosine-based activation motifs (ITAMs) that transmit activation signals to T cells following binding of the cognate antigen. In many cases, CD3ζ provides the primary T cell activation signal, but not the full-blown activation signal, which requires co-stimulatory signaling.

[0107] In some embodiments, the anti-GPC3 CAR polypeptide disclosed herein may further include one or more costimulatory signaling domains. For example, the costimulatory domains of CD28 and / or 4-1BB can be used to transmit complete proliferation / survival signals and the main signaling mediated by CD3ζ. In some examples, the CAR disclosed herein includes CD28 costimulatory molecules. In other examples, the CAR disclosed herein includes 4-1BB costimulatory molecules. In some embodiments, CAR includes a CD3ζ signaling domain and a CD28 costimulatory domain. In other embodiments, CAR includes a CD3ζ signaling domain and a 4-1BB costimulatory domain. In yet other embodiments, CAR includes a CD3ζ signaling domain, a CD28 costimulatory domain, and a 4-1BB costimulatory domain.

[0108] Table 1 provides examples of signaling domains derived from 4-1BB, CD28, and CD3ζ that can be used herein.

[0109] (v) Exemplary anti-GPC3 CAR polypeptides

[0110] Exemplary anti-GPC3 CAR polypeptides are provided in Table 1 below, all of which are within the scope of the present disclosure (including both mature anti-GPC3 CAR, i.e., without an N-terminal signal peptide, and precursor anti-GPC3 CAR, i.e., with an N-terminal signal peptide). Nucleic acids encoding any anti-GPC3 CAR construct disclosed herein are also within the scope of the present disclosure, such as those disclosed in Table 1. The nucleic acid may be located in a suitable vector, such as 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.

[0111] C. Methods for preparing genetically engineered T cells

[0112] The genetically engineered T cells disclosed herein can be prepared by genetic editing of parental T cells or their precursor cells by conventional gene editing methods or those described herein.

[0113] (a) T cells

[0114] In some embodiments, T cells can be derived from one or more suitable mammals, for example, one or more human donors. T cells can be obtained from a variety of sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus, infection site tissue, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments, a variety of techniques known to those skilled in the art can be used, such as sedimentation, for example FICOLL TM Isolate and obtain T cells from a unit of blood collected from a subject.

[0115] In some instances, T cells can be separated from a mixture of immune cells (such as those described herein) to produce isolated T cell populations. For example, after isolating peripheral blood mononuclear cells (PBMCs), before or after activation, amplification and / or genetic modification, cytotoxic T lymphocytes and helper T lymphocytes can be classified into naive T cell subsets, memory T cell subsets, and effector T cell subsets.

[0116] Specific subpopulations of T cells expressing one or more of the following cell surface markers can be further separated by positive or negative selection techniques: TCRαβ, CD3, CD4, CD8, CD27CD28, CD38CD45RA, CD45RO, CD62L, CD127, CD122, CD95, CD197, CCR7, KLRG1, MCH-I protein and / or MCH-II protein. In some embodiments, specific T cell subpopulations expressing one or more markers selected from TCRab, CD4 and / or CD8 are further separated by positive or negative selection techniques. In some embodiments, T cell subpopulations can be separated by positive selection or negative selection before and / or after genetic engineering.

[0117] The isolated T cell population may express one or more T cell markers, including but not limited to CD3+, CD4+, CD8+, or a combination thereof. In some embodiments, T cells are isolated from a donor or subject and first activated and stimulated in vitro to proliferate before gene editing.

[0118] In some cases, the T cell population comprises primary T cells isolated from one or more human donors. Such T cells are terminally differentiated, untransformed, dependent on cytokines and / or growth factors for growth, and / or have a stable genome.

[0119] Alternatively, T cells can be derived from stem cells (eg, HSCs or iPSCs) by in vitro differentiation.

[0120] T cells from a suitable source may undergo one or more rounds of stimulation, activation and / or expansion. T cells may generally be activated and expanded using methods such as those described in U.S. Pat. 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, 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 introducing the genome editing composition into the T cells.

[0121] In some embodiments, before the gene editing composition is introduced into the T cell, the T cell is 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. In some embodiments, the T cell is activated while the genome editing composition is introduced into the T cell. In some cases, the T cell group can be expanded and / or activated after genetic editing as disclosed herein. T cell groups or separated T cells generated by any gene editing method described herein are also within the scope of the present disclosure.

[0122] (b) Gene Editing Methods

[0123] Conventional gene editing methods or those described herein can be used to prepare any genetically engineered T cells to edit one or more target genes disclosed herein (targeted editing). Targeted editing can be achieved by a method that is independent of nucleases or by a method that is dependent on nucleases. In the targeted editing method that is independent of nucleases, homologous recombination is guided by homologous sequences flanking exogenous polynucleotides through the enzymatic mechanism of the host cell to introduce endogenous sequences. Exogenous polynucleotides can introduce deletions, insertions or replacements of nucleotides in endogenous sequences.

[0124] Alternatively, nuclease-dependent methods can achieve higher frequencies of targeted editing by specifically introducing double-strand breaks (DSBs) through specific rare-cutting nucleases (e.g., endonucleases). Such targeted editing that relies on nucleases also utilizes DNA repair mechanisms, such as non-homologous end joining (NHEJ) that occurs in response to DSBs. DNA repair performed by NHEJ typically results in random insertions or deletions (indels) of a small amount of endogenous nucleotides. In contrast to NHEJ-mediated repair, repair can also be performed by homology-directed repair (HDR). When there is a donor template containing exogenous genetic material with a pair of homologous arms on both sides, the exogenous genetic material can be introduced into the genome by HDR, which results in targeted integration of the exogenous genetic material.

[0125] In some embodiments, gene disruption can be performed by deleting genomic sequences using two guide RNAs. Methods for generating genomic deletions in cells (e.g., knocking out genes in cells) using CRISPR-Cas gene editing technology are known (Bauer DE et al. Vis. Exp. 2015; 95: e52118).

[0126] 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).

[0127] In addition, the DICE (Dual Integrase Cassette Exchange) system using phiC31 and Bxb1 integrases can also be used for targeted integration. Some exemplary methods are disclosed in detail below.

[0128] CRISPR-Cas9 gene editing system

[0129] The CRISPR-Cas9 system is a naturally occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided DNA-targeting platform for gene editing. It relies on the DNA nuclease Cas9 and two noncoding RNAs, crisprRNA (crRNA) and trans-activating RNA (tracrRNA) to target DNA cleavage. CRISPR, short for Clustered Regularly Interspaced Short Palindromic Repeats, is a family of DNA sequences found in the genomes of bacteria and archaea that contain DNA fragments (spacer DNA) similar to foreign DNA previously exposed to the cell, such as by viruses that have already infected or attacked prokaryotes. Prokaryotes use these DNA fragments to detect and destroy similar foreign DNA upon reintroduction, such as DNA from similar viruses during subsequent attacks. Transcription of the CRISPR locus results in the formation of an RNA molecule containing the spacer sequence, which binds and targets the Cas (CRISPR-associated) protein that is able to recognize and cleave foreign, exogenous DNA. Many types and classes of CRISPR / Cas systems have been described (see, e.g., Koonin et al., (2017) Curr Opin Microbiol 37:67-78).

[0130] The crRNA typically pairs with a 20-nucleotide (nt) sequence in the target DNA via Watson-Crick base pairing to drive recognition and specificity of the CRISPR-Cas9 complex. Changing the sequence of the 5' 20nt in the crRNA allows the CRISPR-Cas9 complex to be targeted to a specific locus. The CRISPR-Cas9 complex only binds to DNA sequences containing a sequence that matches the first 20nt of the crRNA, provided that the target sequence is followed by a specific short DNA motif (with the sequence NGG), called the protospacer adjacent motif (PAM).

[0131] The tracrRNA hybridizes with the 3' end of the crRNA to form an RNA duplex structure, which binds to the Cas9 endonuclease to form a catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA.

[0132] Once the CRISPR-Cas9 complex binds to the 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, generating a double-strand break (DSB) in which both strands of the DNA terminate with base pairs (blunt ends).

[0133] After the CRISPR-Cas9 complex binds to DNA at a specific target site and forms a site-specific DSB, the next critical step is to repair the DSB. Cells use two major DNA repair pathways to repair DSBs: non-homologous end joining (NHEJ) and homology-directed repair (HDR).

[0134] NHEJ is a powerful repair mechanism that is highly active in most cell types, including non-dividing cells. NHEJ is error-prone and usually results in the removal or addition of 1 to hundreds of nucleotides at the DSB site, although such modifications are usually less than 20nt. The resulting insertions and deletions (indels) may damage the coding or non-coding regions of the gene. Alternatively, HDR uses long extended homologous donor DNA provided by endogenous or exogenous sources 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 used as a repair operator.

[0135] Endonucleases for CRISPR

[0136] In some embodiments, Cas9 (CRISPR-associated protein 9) endonucleases are used in CRISPR methods to prepare genetically engineered T cells disclosed herein. The Cas9 enzyme can be an enzyme from Streptococcus pyogenes, but other Cas9 homologs can also be used. It should be understood that wild-type Cas9 or a modified version of Cas9 (e.g., an evolved version of Cas9, or a Cas9 ortholog or variant) can be used, as provided herein. In some embodiments, Cas9 can be replaced by another RNA-guided endonuclease, such as Cpf1 (Class II CRISPR / Cas system).

[0137] In some embodiments, the CRISPR / Cas system comprises components derived from type I, type II, or type III systems. An updated classification scheme for CRISPR / Cas loci defines class 1 and class 2 CRISPR / Cas systems, with types I to 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". Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. Cpf1 nuclease (Zetsche et al., (2015) Cell 163:1-13) is homologous to Cas9 and contains a RuvC-like nuclease domain.

[0138] In some embodiments, the Cas nuclease is from a type II CRISPR / Cas system (e.g., a Cas9 protein from a CRISPR / Cas9 system). In some embodiments, the Cas nuclease is from a class 2 CRISPR / Cas system (a single protein Cas nuclease, such as a Cas9 protein or a Cpf1 protein). The Cas9 and Cpf1 protein families are enzymes with DNA endonuclease activity and can be guided to cleave a desired nucleic acid target by designing appropriate guide RNAs, as further described herein.

[0139] In some embodiments, the Cas nuclease may include more than one nuclease domain. For example, the Cas9 nuclease may include at least one RuvC-like nuclease domain (e.g., Cpfl) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease introduces DSB in the target sequence. In some embodiments, the Cas9 nuclease is modified to contain only one functional nuclease domain. For example, the Cas9 nuclease is modified so that one of the nuclease domains is mutated or completely or partially missing to reduce its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease is modified to contain no functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease is modified to contain no functional HNH-like nuclease domain. In some embodiments where only one nuclease domain is functional, the Cas9 nuclease is a nickase capable of introducing a single-strand break ("nick") into the target sequence. In some embodiments, the conservative amino acids in the Cas9 nuclease domain are replaced to reduce or change the nuclease activity. In some embodiments, the Cas nuclease nickase comprises an amino acid substitution in a RuvC-like nuclease domain. An exemplary amino acid substitution in a RuvC-like nuclease domain comprises D10A (based on Streptococcus pyogenes Cas9 nuclease). In some embodiments, a nickase comprises an amino acid substitution in a HNH-like nuclease domain. An exemplary amino acid substitution in a HNH-like nuclease domain comprises E762A, H840A, N863A, H983A, and D986A (based on Streptococcus pyogenes Cas9 nuclease). Table 2 below provides an example (SEQ ID NO: 70).

[0140] In some embodiments, the Cas nuclease is from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease is a component of the Cascade complex of a 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.

[0141] Guide RNA (gRNA)

[0142] CRISPR technology involves the use of genome targeting nucleic acids that can locate endonucleases to specific target sequences within a target gene, thereby performing gene editing on a specific target sequence. The nucleic acid targeting the genome can be RNA. The RNA targeting the genome is referred to herein as "guide RNA" or "gRNA". The guide RNA at least comprises a spacer sequence and a CRISPR repeat sequence that hybridizes with a target nucleic acid sequence within a target gene for editing.

[0143] In the type II system, the gRNA also includes a second RNA called a tracrRNA sequence. In the type II gRNA, the CRISPR repeat sequence and the tracrRNA sequence hybridize to each other to form a duplex. In the type V gRNA, the crRNA forms a duplex. In these two systems, the duplex binds to the site-directed polypeptide so that the guide RNA and the site-directed polypeptide form a complex. In some embodiments, the genome targeting nucleic acid provides target specificity for the complex by virtue of its binding to the site-directed polypeptide. Therefore, the nucleic acid targeting the genome guides the activity of the site-directed polypeptide.

[0144] As will be appreciated by one of ordinary skill in the art, each guide RNA is designed to contain 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).

[0145] In some embodiments, the nucleic acid (e.g., gRNA) targeting a genome is a double-molecule guide RNA. In some embodiments, the nucleic acid (e.g., gRNA) targeting a genome is a single-molecule guide RNA.

[0146] The bimolecular guide RNA comprises two RNA molecular chains. The first chain comprises an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence in the 5' to 3' direction. The second chain comprises a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' end tracrRNA sequence, and an optional tracrRNA extension sequence.

[0147] The single-molecule guide RNA (referred to as "sgRNA") in the II-type system comprises an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single-molecule guide adapter, a minimal tracrRNA sequence, a 3' end tracrRNA sequence, and an optional tracrRNA extension sequence in the 5' to 3' direction. The optional tracrRNA extension may include elements that contribute additional functions (e.g., stability) to the guide RNA. The single-molecule guide adapter connects the minimal CRISPR repeat and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension comprises one or more hairpin structures. The single-molecule guide RNA in the V-type system comprises a minimal CRISPR repeat sequence and a spacer sequence in the 5' to 3' direction.

[0148] The spacer sequence in the gRNA is a sequence (e.g., a 20 nucleotide sequence) that defines a target sequence (e.g., a DNA target sequence, such as a genomic target sequence) of a target gene of interest. In some embodiments, the range of the spacer sequence is 15 to 30 nucleotides. For example, the spacer sequence can contain 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the spacer sequence comprises 20 nucleotides. The "target sequence" is located in a target gene adjacent to a PAM sequence and is a sequence to be modified by a nuclease (e.g., Cas9) guided by RNA.

[0149] "Target sequence" is located on the so-called PAM strand in "target nucleic acid", and target nucleic acid is a double-stranded molecule containing a PAM strand and a complementary non-PAM strand. Those skilled in the art recognize that the gRNA spacer sequence hybridizes with the 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, if 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 gRNA interacts with the target nucleic acid of interest in a sequence-specific manner by hybridization (i.e., base pairing). Therefore, the nucleotide sequence of the spacer varies according to the target sequence of the target nucleic acid of interest.

[0150] In the CRISPR / Cas system of this article, the spacer sequence is designed to hybridize with the 5' end region of the target nucleic acid located at the PAM that can be recognized by the Cas9 enzyme used in the system. The spacer may be a perfect match to the target sequence or it may have mismatches. Each Cas9 enzyme has a specific PAM sequence that is recognized in the target DNA. For example, Streptococcus pyogenes (S.pyogenes) recognizes a PAM in the target nucleic acid that contains the sequence 5'-NRG-3', where R contains A or G, where N is any nucleotide, and N is adjacent to the 3' end of the target nucleic acid sequence targeted by the spacer sequence.

[0151] 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 5' of the first nucleotide immediately adjacent to the PAM. For example, in a sequence comprising 5'-NNNNNNNNNNNNNNNNNNNN NRG -3' sequence, the target nucleic acid can be a sequence corresponding to N, wherein N can be any nucleotide, and the underlined NRG sequence is Streptococcus pyogenes PAM.

[0152] The guide RNA disclosed herein can target any sequence of interest through the spacer sequence in the crRNA. In some embodiments, the degree of complementarity between the spacer sequence of the guide RNA and the target sequence in 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 is 100% complementary to the target sequence in the target gene. In other embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene can contain up to 10 mismatches, such as 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.

[0153] For any gRNA sequences provided herein, those sequences not explicitly indicating modification are intended to encompass both the unmodified sequence and sequences with any suitable modification.

[0154] The length of the spacer sequence in any gRNA disclosed herein can depend on the CRISPR / Cas9 system and components for editing any target gene also disclosed herein. For example, different Cas9 proteins from different bacterial species have different optimal spacer sequence lengths. Therefore, the length of 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 nucleotides or more than 50 nucleotides. In some embodiments, the length of the spacer sequence can be 18-24 nucleotides. In some embodiments, the length of the targeting sequence can be 19-21 nucleotides. In some embodiments, the spacer sequence can include 20 nucleotides in length.

[0155] In some embodiments, the gRNA may be an sgRNA that may include a spacer sequence of 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA may include a spacer sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA may include a spacer sequence of more than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA includes a spacer sequence of a variable length of 17-30 nucleotides at the 5' end of the sgRNA sequence. Table 2 below provides examples. In these exemplary sequences, the "n" segment refers to the spacer sequence at the 5' end.

[0156] In some embodiments, the sgRNA does not include uracil at the 3' end of the sgRNA sequence. In other embodiments, the sgRNA can include one or more uracils at the 3' end of the sgRNA sequence. For example, the sgRNA can include 1-8 uracil residues at the 3' end of the sgRNA sequence, such as 1, 2, 3, 4, 5, 6, 7, or 8 uracil residues at the 3' end of the sgRNA sequence.

[0157] Any gRNA disclosed herein, including any sgRNA, may be unmodified. Alternatively, it may contain one or more modified nucleotides and / or a modified backbone. For example, a modified gRNA such as an sgRNA may contain one or more 2'-O-methyl phosphorothioate nucleotides, which may be located at the 5' end, the 3' end, or both.

[0158] In certain embodiments, more than one guide RNA can be used with the CRISPR / Cas nuclease system. Each guide RNA can contain a different targeting sequence so that the CRISPR / Cas system cuts more than one target nucleic acid. In some embodiments, one or more guide RNAs can have the same or different properties, such as activity or stability within the Cas9RNP complex. When more than one guide RNA is used, each guide RNA can be encoded on the same or different vectors. The promoter used to drive the expression of more than one guide RNA is the same or different.

[0159] In some embodiments, the gRNA disclosed herein targets the Reg1 gene, for example, targets a site within exon 1, exon 2, exon 3, exon 4, exon 5, or exon 6 of the Reg1 gene. Such a gRNA may comprise a spacer sequence that is complementary (completely or partially) to a target sequence in exon 2 or exon 4 of the Reg1 gene, or a fragment thereof. Exemplary target sequences and exemplary gRNA sequences for Reg1 are provided in Table 2 below.

[0160] In some embodiments, the gRNA disclosed herein targets a TGFBRII gene, for example, a site within exon 1, exon 2, exon 3, exon 4, exon 5, or exon 6 of the TGFBRII gene. Such a gRNA may comprise a spacer sequence complementary (completely or partially) to a target sequence in exon 4 or exon 5 of the TGFBRII gene, or a fragment thereof. Exemplary target sequences and exemplary gRNA sequences for TGFBRII are provided in Table 2 below.

[0161] In some embodiments, the gRNA disclosed herein targets the cbl-b gene, for example, a site within exon 1, exon 2, exon 7, exon 9, exon 11, or exon 12 of the cbl-b gene. Such a gRNA may include a spacer sequence complementary (completely or partially) to a target sequence in exon 2 of a cbl-b gene or a fragment thereof. In other examples, the gRNA may include a spacer sequence complementary (completely or partially) to a target sequence in exon 7 of a cbl-b gene or a fragment thereof. Alternatively, the gRNA may include a spacer sequence complementary (completely or partially) to a target sequence in exon 9 of a cbl-b gene or a fragment thereof. Exemplary target sequences and exemplary gRNA sequences of cbl-b genes are provided in Table 2 below.

[0162] In some embodiments, the gRNA disclosed herein targets the β2M gene, for example, a suitable site within the β2M gene. See also WO2019097305, for the purposes and themes cited herein, the relevant disclosures thereof are incorporated herein by reference. Other gRNA sequences can 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 produce a break in the β2M genomic region, resulting in Indel in the β2M gene destroying the expression of mRNA or protein. Table 2 below provides exemplary spacer sequences and gRNA targeting the β2M gene.

[0163] In some embodiments, the gRNA disclosed herein targets the TRAC gene. See also WO2019097305, for the purposes and themes cited herein, the relevant disclosures of which are incorporated herein by reference. The TRAC gene sequence located on chromosome 14 can be used to design other gRNA sequences (GRCh38: chromosome 14: 22,547,506-22,552,154; Ensembl; ENSG00000277734). In some embodiments, the gRNA and RNA-guided nuclease targeting the TRAC genomic region produce breaks in the TRAC genomic region, resulting in Indel in the TRAC gene destroying the expression of mRNA or protein. Table 2 below provides exemplary spacer sequences and gRNA targeting the TRAC gene.

[0164] For example, guide RNA or other smaller RNA used in the CRISPR / Cas / Cpf1 system can be easily synthesized by chemical methods, as shown below and described in the art. Although chemical synthesis procedures continue to expand, as the length of polynucleotides increases significantly beyond a hundred or so nucleotides, purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, avoiding the use of gels such as PAGE) tends to become more challenging. One method for generating longer RNAs is to produce two or more molecules linked together. Longer RNAs, such as RNAs encoding Cas9 or Cpf1 endonucleases, are more easily generated enzymatically. Various types of RNA modifications can be introduced during or after chemical synthesis and / or enzymatic generation of RNA, for example, modifications that enhance stability, reduce the likelihood or extent of innate immune responses, and / or enhance other properties, as described in the art.

[0165] In some examples, the gRNA of the present disclosure can be produced by in vitro transcription (IVT), synthesis and / or chemical synthesis methods or a combination thereof. Enzymatic (IVT), solid phase, liquid phase, combinatorial synthesis methods, small area synthesis and ligation methods are used. In one embodiment, the gRNA is prepared using an IVT enzymatic synthesis method. Methods for preparing polynucleotides by IVT are known in the art and are described in WO2013 / 151666. Therefore, the present disclosure also includes polynucleotides, such as DNA, constructs and vectors, for in vitro transcription of the gRNA described herein.

[0166] Various types of RNA modifications can be introduced during or after the chemical synthesis and / or enzymatic generation of RNA, for example, to enhance stability, reduce the possibility or degree of innate immune response, and / or enhance the modification of other properties, as described in the art. In some embodiments, non-natural modified nucleobases can be introduced into any gRNA disclosed herein during or after synthesis. In certain embodiments, the modification is on internucleoside linkages, purine or pyrimidine bases, or sugars. In some embodiments, the modification is introduced at the end of the gRNA by chemical synthesis or by a polymerase. Examples of modified nucleic acids and their synthesis are disclosed in WO2013 / 052523. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol.76, 99-134 (1998).

[0167] In some embodiments, enzymatic or chemical ligation methods can be used to conjugate polynucleotides or regions thereof to various functional moieties, such as targeting or delivery agents, fluorescent markers, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).

[0168] In some embodiments of the present disclosure, the CRISPR / Cas nuclease system for genetically editing any one of the target genes disclosed herein may include at least one guide RNA. In some examples, the CRISPR / Cas nuclease system may contain multiple gRNAs, such as 2, 3 or 4 gRNAs. Such multiple gRNAs can target different sites in the same target gene. Alternatively, multiple gRNAs can target different genes. In some embodiments, the guide RNA and the Cas protein can form a ribonucleoprotein (RNP), such as a CRISPR / Cas complex. The guide RNA can guide the Cas protein to a target sequence on one or more target genes as disclosed herein, wherein the Cas protein cuts the target gene at the target site. In some embodiments, the CRISPR / Cas complex is a Cpf1 / guide RNA complex. In some embodiments, the CRISPR complex is a type II CRISPR / Cas9 complex. In some embodiments, the Cas protein is a Cas9 protein. In some embodiments, the CRISPR / Cas9 complex is a Cas9 / guide RNA complex.

[0169] In some embodiments, TIDE analysis can be used to determine the insertion and deletion frequency (editing frequency) of a specific CRISPR / Cas nuclease system comprising one or more specific gRNAs, and TIDE analysis can be used to identify efficient gRNA molecules for editing target genes. In some embodiments, efficient gRNA produces a gene editing frequency higher than 80%. For example, if the gRNA produces a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%, the gRNA is considered to be efficient.

[0170] Delivery of guide RNA and nuclease to T cells

[0171] The CRISPR / Cas nuclease system disclosed herein, comprising one or more gRNA and at least one RNA-guided nuclease, optionally as a donor template disclosed below, can be delivered to a target cell (e.g., T cell) for genetic editing of a target gene by conventional methods. In some embodiments, the components of the CRISPR / Cas nuclease system disclosed herein can be delivered to the target cell alone simultaneously or sequentially. In other embodiments, the components of the CRISPR / Cas nuclease system can be delivered together to the target, for example as a complex. In some cases, the gRNA and RNA-guided nuclease can be pre-compounded together to form a ribonucleoprotein (RNP), which can then be delivered to the target cell.

[0172] RNP is useful for gene editing, at least because it can minimize the risk of promiscuous interactions in a cell environment rich in nucleic acids and protect RNA from degradation. The method of forming RNP is known in the art. In some embodiments, the RNP containing RNA-guided nuclease (e.g., 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, RNP can be delivered to T cells by electroporation.

[0173] In some embodiments, the RNA-guided nuclease can be delivered to the cell in a DNA vector that expresses the RNA-guided nuclease in the cell. In other examples, the RNA-guided nuclease can be delivered to the cell in the form of RNA encoding the RNA-guided nuclease and expressed in the cell. Alternatively or in addition, the gRNA targeting a gene can be delivered to the cell as RNA or a DNA vector that expresses the gRNA in the cell.

[0174] The delivery of RNA-guided nuclease, gRNA and / or RNP can be by direct injection or using known methods to transfect cells, such as electroporation or chemical transfection. Other cell transfection methods can be used. In some cases, the Cas9 enzyme can form a kind of RNP with all gRNAs targeting TRAC gene, β2M gene, Reg1 gene and TGFBRII gene, and is delivered to T cells by an electroporation event. Alternatively, the Cas9 enzyme can form two or more RNPs, which together contain all gRNAs targeting TRAC gene, β2M gene, Reg1 gene and TGFBRII gene. Multiple RNPs can be delivered to T cells by continuous electroporation events, such as two continuous electroporation events.

[0175] In other embodiments, viral vectors such as one or more lentiviral vectors can be used to deliver nucleic acids encoding nucleases and optionally one or more gRNAs to target cells (e.g., T cells) for genetic editing of one or more target genes disclosed herein.

[0176] Other gene editing methods

[0177] In addition to the CRISPR methods disclosed herein, other gene editing methods known in the art can also be used to prepare the genetically engineered T cells disclosed herein. Some examples include gene editing methods involving zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), restriction endonucleases, meganucleases, homing endonucleases, etc.

[0178] ZFN is a targeted nuclease comprising a nuclease fused to a zinc finger DNA binding domain (ZFBD), which is a polypeptide domain that binds DNA in a sequence-specific manner through one or more zinc fingers. A zinc finger is a domain of about 30 amino acids within a zinc finger binding domain, the structure of which is stabilized by the coordination of zinc ions. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. The designed zinc finger domain is a domain that does not exist in nature, and its design / composition is mainly derived from rational criteria, for example, applying substitution rules and computerized algorithms to process information in databases storing existing ZFP design and binding data information. See, for example, U.S. Patent Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98 / 53058; WO 98 / 53059; WO98 / 53060; WO 02 / 016536 and WO 03 / 016496. The selected zinc finger domain is a domain not found in nature and is generated primarily from empirical processes such as phage display, interaction capture or hybrid selection. ZFNs are described in more detail in U.S. Pat. No. 7,888,121 and U.S. Pat. No. 7,972,854. The most recognized example of a ZFN is the fusion of the FokI nuclease with a zinc finger DNA binding domain.

[0179] TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain. A "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 responsible for the binding of the TAL effector protein to DNA. TAL effector proteins are secreted by Xanthomonas plant pathogens during infection. These proteins enter the nucleus of plant cells, bind effector-specific DNA sequences through their DNA binding domains, and activate gene transcription on these sequences through their transactivation domains. The specificity of the TAL effector DNA binding domain depends on the imperfect 34 amino acid repeat sequences of the effector variable number, which contain polymorphisms at selected repeat positions, called repeat variable-residues (RVDs). TALEN is described in more detail in U.S. Patent Application No. 2011 / 0145940. The most recognized TALEN example in the art is a fusion polypeptide of the FokI nuclease and the TAL effector DNA binding domain.

[0180] Additional examples of targeted nucleases suitable for use herein include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and Wβ / SPBc / TP901-1, whether used alone or in combination.

[0181] Any of the nucleases disclosed herein, including CRISPR / Cas nucleases, can be delivered using vector systems including, but not limited to, plasmid vectors, DNA minicircles, retroviral vectors, lentiviral vectors, adenoviral vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, and combinations thereof.

[0182] Gene transfer methods based on conventional viruses and non-viruses can be used to introduce nucleic acids encoding nucleases and donor templates into cells (e.g., T cells). Non-viral vector delivery systems include DNA plasmids, DNA minicircles, naked nucleic acids, and nucleic acids compounded with delivery vectors such as liposomes or poloxamer. Viral vector delivery systems include DNA and RNA viruses that have free or integrated genomes after delivery to cells.

[0183] Non-viral delivery methods for nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, naked RNA, capped RNA, artificial virions, and drug-enhanced DNA uptake. Acoustic perforation using, for example, a Sonitron 2000 system (Rich-Mar) can also be used to deliver nucleic acids. Some specific examples are provided below.

[0184] D. Delivery of anti-GPC3 CAR constructs to T cells

[0185] In some embodiments, the nucleic acid encoding anti-GPC3CAR can be introduced into any genetically engineered T cell disclosed herein by methods known to those skilled in the art. For example, the coding sequence of anti-GPC3CAR can be cloned into a vector, which can be introduced into genetically engineered T cells to express anti-GPC3CAR. A variety of different methods are known in the art that can be used to introduce any nucleic acid or expression vector disclosed herein into immune effector cells. Non-limiting examples of methods for introducing nucleic acids into cells include: lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, phototransfection, particle-based transfection (e.g., nanoparticle transfection)), or transfection using liposomes (e.g., cationic liposomes), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, puncture transfection, hydrodynamic delivery, gene gun, magnetic transfection, viral transfection, and nuclear transfection.

[0186] In some examples, adeno-associated virus (AAV) can be used to deliver nucleic acid encoding anti-GPC3 CAR constructs to cells. AAV is a small virus that can be site-specifically integrated into the host genome, so transgenes such as anti-GPC3 CAR can be delivered. Inverted terminal repeats (ITRs) are present on the flanks of the AAV genome and / or transgenes of interest and serve as a replication origin. There are also rep and cap proteins in the AAV genome, which form capsids when transcribed, and the capsids encapsulate the AAV genome for delivery to target cells. The surface receptors on these capsids give the AAV serotype, which determines which target organs the capsid will mainly bind to, thereby determining which cells AAV will infect most effectively. There are 12 currently known human AAV serotypes. In some embodiments, the AAV used to deliver anti-GPC3 CAR encoding nucleic acids is AAV serotype 6 (AAV6).

[0187] Adeno-associated virus is one of the most commonly used viruses in gene therapy for a variety of reasons. First, AAV does not cause an immune response after administration to mammals (including humans). Second, AAV can be effectively delivered to target cells, especially when considering the selection of the appropriate AAV serotype. Finally, AAV is able to infect both dividing and non-dividing cells because the genome can persist in host cells without the need for integration. This property makes them ideal candidates for gene therapy.

[0188] The nucleic acid encoding the CAR can be designed to be inserted into a genomic site of interest in a host T cell. In some embodiments, the target genomic site can be located in a safe harbor locus.

[0189] In some embodiments, the nucleic acid encoding the anti-GPC3 CAR (e.g., by a donor template, which can be carried by a viral vector such as an adeno-associated virus (AAV) vector) can be designed so that it can be inserted into the position of the TRAC gene to destroy the TRAC gene in the genetically engineered T cells and express the CAR polypeptide. Destroying TRAC can lead to loss of endogenous TCR function. For example, a nuclease (such as those described herein) and one or more gRNAs targeting one or more TRAC genomic regions can be used to produce a disruption in the TRAC gene. Any gRNA specific to the TRAC gene and target region disclosed herein can be used for this purpose.

[0190] In some examples, genomic deletions in the TRAC gene and replacement of the anti-GPC3 CAR coding segment can be generated by homology directed repair or HDR (e.g., using a donor template, which can be part of a viral vector such as an adeno-associated virus (AAV) vector). In some embodiments, disruptions in the TRAC gene and insertion of the anti-GPC3 CAR coding segment can be generated using endonucleases such as those disclosed herein and one or more gRNAs targeting one or more TRAC genomic regions.

[0191] The donor template disclosed herein may contain the coding sequence of anti-GPC3 CAR. In some instances, the flank of the anti-GPC3CAR coding sequence may be two homologous regions, to allow effective HDR to be performed at the genomic position of interest (e.g., at the TRAC gene) using gene editing methods known in the art. In some instances, a CRISPR-based method may be used. In this case, both DNA chains at the target locus can be cut by the CRISPR Cas9 enzyme guided by the gRNA specific to the target locus. Then HDR occurs to repair double-strand breaks (DSB) and insert the donor DNA encoding CAR. In order to correctly achieve this, the donor sequence is designed to have flanking residues that are complementary to the sequences around the DSB site in the target gene (such as the TRAC gene) (hereinafter "homology arms"). These homology arms serve as templates for DSB repair and allow HDR to become a substantially error-free mechanism. The rate of homology-directed repair (HDR) is a function of the distance between the mutation and the cleavage site, so it is important to select overlapping or nearby target sites. The template may contain additional sequences flanked by regions of homology or may contain sequences that differ from the genomic sequence, thereby allowing sequence editing.

[0192] Alternatively, the donor template may not have regions of homology to the target location in DNA and may be integrated by NHEJ-dependent end joining at the target site following cleavage.

[0193] The donor template can be DNA or RNA, single-stranded and / or double-stranded, and can be introduced into the cell in a linear or circular form. If introduced in a linear form, the end of the donor sequence can be protected by methods known to those skilled in the art (e.g., from exonucleolytic degradation). For example, one or more dideoxynucleotide residues are added to the 3' end of the linear molecule and / or a self-complementary oligonucleotide is connected 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. Other methods for protecting exogenous polynucleotides from degradation include, but are not limited to, adding terminal amino groups and using modified internucleotide bonds, such as phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.

[0194] The donor template can be introduced into the cell as part of a vector molecule having additional sequences such as, for example, an origin of replication, a promoter, and genes encoding antibiotic resistance. In addition, the donor template can be introduced into the cell as naked nucleic acid, as a nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by a virus (e.g., adenovirus, AAV, herpes virus, retrovirus, lentivirus, and integrase-deficient lentivirus (IDLV)).

[0195] In some embodiments, the donor template can be inserted into a site near the endogenous promoter (e.g., downstream or upstream), so that its expression can be driven by the endogenous promoter. In other embodiments, the donor template may include an exogenous promoter and / or enhancer, such as a constitutive promoter, an inducible promoter, or a tissue-specific promoter to control the expression of the CAR gene. In some embodiments, the exogenous promoter is an EF1α promoter, see, for example, SEQ ID NO: 72, as shown in Table 3 below. Other promoters may be used.

[0196] In addition, the exogenous sequence may also contain transcriptional or translational regulatory sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and / or polyadenylation signals.

[0197] In some embodiments, the donor template for delivering anti-GPC3 CAR can be an AAV vector inserted with a nucleic acid fragment, the nucleic acid fragment comprising the coding sequence of anti-GPC3 CAR and optionally a regulatory sequence for expressing anti-GPC3 CAR (e.g., a promoter such as the EF1a promoter provided in Table 3), which can be flanked by homology arms for inserting the coding sequence and the regulatory sequence into the genomic locus of interest. 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 in the TRAC gene, such as a fragment comprising the nucleotide sequence of SEQ ID NO: 58. In some specific examples, the donor template for delivering anti-GPC3 CAR can include the nucleic acid sequence shown in SEQ ID NO: 12, 75, 27, 30, 33 or 36, which can be flanked by upstream and downstream homology arms (e.g., SEQ ID NO: 71 and SEQ ID NO: 74). In some cases, a nucleic acid encoding a CAR can be inserted into a disrupted TRAC gene, for example replacing a fragment of SEQ ID NO:58.

[0198] In other examples, a lentiviral vector can be used to deliver nucleic acids encoding anti-GPC3 CAR constructs to cells. Lentiviral vectors can infect dividing cells and non-dividing cells. Therefore, they can effectively deliver transgenes to non-proliferating or slowly proliferating cells, which makes them attractive in clinical applications. A lentiviral vector carrying a nucleic acid encoding any anti-GPC3 CAR disclosed herein can be constructed according to conventional methods. See also Example 5 below.

[0199] E. Exemplary anti-GPC3 CAR-T cells optionally with multiple genetic edits

[0200] In some embodiments, the genetically engineered T cell population disclosed herein expresses an anti-GPC3 CAR as disclosed herein (e.g., those provided in Table 1). Such genetically engineered T cells may also comprise a disrupted TRAC gene, a disrupted β2M gene, a disrupted Reg1 gene, a disrupted TGFBRII gene, a disrupted cbl-b gene, or a combination thereof). The nucleotide sequence encoding the anti-GPC3 CAR may be inserted into a genetic site of interest, e.g., into a disrupted TRAC gene (e.g., replacing the site targeted by the sgRNA listed in Table 2 below).

[0201] In some examples, the genetically engineered T cell groups disclosed herein express anti-GPC3 CAR as disclosed herein (e.g., those provided in Table 1) and include a disrupted TRAC gene and a disrupted β2M gene. The nucleotide sequence encoding anti-GPC3 CAR can be inserted into a genetic locus of interest, for example, in a disrupted TRAC gene (e.g., replacing the site targeted by the sgRNA listed in Table 2 below). Such genetically engineered T cell groups may further include a disrupted Reg1 gene, a disrupted TGFBRII gene, a disrupted cbl-b gene, or a combination thereof. For example, a genetically engineered T cell group may further include a disrupted Reg1 gene and a disrupted TGFBRII gene. Alternatively, a genetically engineered T cell group may further include a disrupted TGFBRII gene and a disrupted cbl-b gene. In other examples, a genetically engineered T cell group may further include a disrupted cbl-b gene. Such genetically engineered T cells may further include additional gene editing, such as a disrupted Reg-1 and / or TGFBRII gene. Alternatively, the genetically engineered T cells may have wild-type Reg-1 and / or TGFBRII genes.

[0202] In some embodiments, the genetically engineered T cell population disclosed herein expresses an anti-GPC3 CAR as disclosed herein (e.g., those provided in Table 1) and comprises a disrupted Reg1 gene, a disrupted TGFBRII gene, a disrupted cbl-b gene, or a combination thereof). In some instances, the genetically engineered T cell population expresses an anti-GPC3 CAR and comprises a disrupted Reg1 gene and a disrupted TGFBRII gene. Alternatively, the genetically engineered T cell population may comprise a disrupted TGFBRII gene and a disrupted cbl-b gene. In other instances, the genetically engineered T cell population may further comprise a disrupted cbl-b gene. Such genetically engineered T cells may further comprise additional gene editing, such as disrupted Reg-1 and / or TGFBRII genes. Alternatively, the genetically engineered T cells may have wild-type Reg-1 and / or TGFBRII genes. In some cases, the genetically engineered T cells may have wild-type TRAC genes, wild-type β2M genes, or both.

[0203] In some examples, the population of genetically engineered T cells can contain about 50%-99% (e.g., about 55% to about 80%) CAR + T cells, and optionally about 90%-99.9% (e.g., about 95% to about 99.7%) TCR - T cells, about 50% to about 90% (e.g., about 60% to about 80%) β2M - T cells, about 50% to about 90% (e.g., about 60% to about 70%) TGFBRII- T cells, about 50% to about 90% (e.g., about 60% to about 70%) Reg1 - T cells, and / or about 50% to about 90% (e.g., about 60% to about 70%) CBLB - In other examples, the genetically engineered T cell population can contain 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%) of Reg1 - In yet other examples, the genetically engineered T cell population can contain 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%) of CBLB - T cells.

[0204] It should be understood that gene disruption encompasses gene modification by 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, thereby substantially reducing or completely eliminating the activity of the encoded gene product. One or more mutations may be located in a non-coding region, such as a promoter region, a regulatory region that regulates transcription or translation; or an intron region. Alternatively, one or more mutations may be located in a coding region (e.g., in an exon). In some cases, the disrupted gene does not express or expresses a substantially reduced level of the encoded protein. In other cases, the disrupted gene expresses the encoded protein in a mutant form, and the protein is either nonfunctional or significantly reduced in activity. In some embodiments, the disrupted gene is a gene that does not encode a functional protein. In some embodiments, cells comprising disrupted genes do not express (e.g., on the cell surface) detectable levels of (e.g., by antibodies, such as by flow cytometry) proteins encoded by the gene. Cells that do not express detectable levels of protein may be referred to as knockout cells. For example, if β2M protein cannot be detected on the cell surface using an antibody that specifically binds to β2M protein, cells with β2M gene editing can be considered to be β2M knockout cells. On the other hand, when surface expression of a surface receptor (e.g., anti-GPC3 CAR) can be detected by conventional methods (e.g., by flow cytometry or immunostaining), the cell is considered to express such a receptor (e.g., anti-GPC3 CAR) positive (+).

[0205] Any of the anti-GPC3 CAR-T cells disclosed herein can be suspended in a cryopreservation solution (e.g., C55) to form a pharmaceutical composition. Cryopreservation solutions for the present disclosure may also include adenosine, dextrose, dextran-40, lactobionic acid, sucrose, mannitol, buffers such as 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 alkalis (e.g. sodium hydroxide, potassium hydroxide, etc.) or combinations thereof. The components of the cryopreservation solution may be dissolved in sterile water (injection quality). Any cryopreservation solution may be substantially free of serum (cannot be detected by conventional methods).

[0206] II. CAR-T cell therapy for GPC3+ cancers

[0207] The anti-GPC3 CAR-T cells disclosed herein can be used to eliminate disease cells expressing GPC3, such as GPC3+ cancer cells. For example, an effective amount of anti-GPC3 CAR-T cells can be administered to a subject in need of treatment by a suitable route (e.g., intravenous infusion).

[0208] The administration step may include placing (e.g., transplanting) anti-GPC3 CAR-T cells into a subject by a method or approach that causes the CAR-T cells to be at least partially located at a desired site (such as a tumor site) so that the desired effect can be produced. Anti-GPC3 CAR-T cells may be administered by any appropriate route, resulting in delivery to a desired location in the subject, wherein at least a portion of the implanted cells or components of the cells remain alive. The survival period of the cells after administration to the subject may be as short as a few hours, such as twenty-four hours, to a few days, to up to several years, or even the life of the subject, i.e., long-term implantation. For example, in some aspects described herein, an effective amount of therapeutic T cells may be administered via a systemic administration route, such as an intraperitoneal or intravenous route.

[0209] In some embodiments, anti-GPC3 CAR-T cells are administered systemically, which refers to administering the cell population indirecty to the target site, tissue, or organ, thereby allowing it to enter the circulatory system of the surrogate subject and thus undergo metabolism and other similar processes. Suitable modes of administration include injection, infusion, infusion, or ingestion. Injection includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous.

[0210] 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 cell can be a subject's own ("self"), that is, the cell is from the same subject. Alternatively, the anti-GPC3 CAR-T cell can be non-self for the subject ("non-self", such as allogeneic, isogenic or xenogeneic). "Allogeneic" refers to that the anti-GPC3 CAR-T cell is not derived from the subject being treated, but from different individuals (donors) of the same species as the subject. The donor is a non-treated individual. The donor is an individual of a non-patient. In some embodiments, the donor is an individual who does not suffer from or is not suspected of suffering from the cancer being treated. In some embodiments, multiple donors, such as two or more donors, are used. In some embodiments, the anti-GPC3 CAR-T cell group administered according to the method described herein comprises allogeneic T cells obtained from one or more donors (e.g., one or more healthy human donors).

[0211] An effective amount refers to the amount of anti-GPC3 CAR-T cells disclosed herein required to prevent or alleviate at least one or more signs or symptoms of a medical condition (e.g., cancer), and relates to a sufficient amount of a composition that provides a desired effect, for example, to treat a subject with a medical condition. An effective amount also includes an amount sufficient to prevent or delay the development of disease symptoms, to change the course of disease symptoms (e.g., but not limited to, to slow the progression of disease symptoms), or to reverse the symptoms of a disease. It should be understood that for any given situation, an appropriate effective amount can be determined by a person of ordinary skill in the art using routine experiments.

[0212] The effect of treatment with the anti-GPC3 CAR-T cells disclosed herein can be determined by a professional clinician. If any or all of the signs or symptoms are changed in a beneficial manner, for example, the functional target level is changed in a beneficial manner (e.g., at least 10% increase), or other clinically accepted disease (e.g., cancer) symptoms or markers are improved or alleviated, then the treatment is considered "effective". Efficacy can also be measured by the subject's lack of deterioration, which can be assessed by hospitalization or the need for medical intervention (e.g., disease progression is prevented or at least slowed). Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of the subject's disease, and includes: (1) inhibiting the disease, such as preventing or slowing the progression of symptoms; (2) alleviating the disease, such as causing symptom regression; and (3) preventing or reducing the likelihood of symptom development.

[0213] In some embodiments, the anti-GPC3 CAR-T cells disclosed herein can be used to eliminate GPC3 in human patients.+ Cancer cells and / or GPC3 treatment + Cancer. In some cases, the human patient may have liver cancer (eg, HCC), gastric cancer, colorectal cancer, lung cancer, ovarian cancer, skin cancer, or thyroid cancer.

[0214] The present disclosure also encompasses combination therapies. For example, the therapeutic T cells disclosed herein can be used together with other therapeutic agents to treat the same indication, or to enhance the efficacy of the therapeutic T cells and / or reduce the side effects of the therapeutic T cells.

[0215] IV. Reagent test kit

[0216] The present disclosure also provides kits for producing genetically engineered T cells, therapeutic T cells and for therapeutic use.

[0217] In some embodiments, the kit provided herein may include components for genetically editing one or more of the TRAC gene, β2M gene, TGFBRII gene, Reg-1 gene and cbl-b gene, and optionally an immune cell population (e.g., leukopak) to which it is genetically edited. Leukopak samples can be enriched leukocyte removal products collected from peripheral blood. It generally contains a variety of blood cells, including monocytes, lymphocytes, platelets, plasma and red blood cells. The components for genetically editing one or more target genes may include suitable endonucleases, such as RNA-guided endonucleases and one or more nucleic acid guides, which guide the cutting of one or more suitable genomic sites by endonucleases. For example, the kit may include Cas enzymes such as Cas9 and one or more gRNAs targeting cbl-b genes. Any gRNA specific to these target genes may be included in the kit. Such a kit may further include components for further gene editing, such as gRNA and optional other endonucleases for editing other target genes such as Reg-1, TGFBRII, cbl-b, β2M and / or TRAC.

[0218] In some embodiments, the kit provided herein may include a genetically engineered T cell population disclosed herein, and one or more components for producing therapeutic T cells also disclosed herein. Such components may include a nucleic acid suitable for gene editing endonucleases and encoding a CAR construct of interest. The nucleic acid encoding CAR may be a part of a donor template disclosed herein, which may contain homology arms flanking the CAR coding sequence. In some cases, the donor template may be carried by a viral vector such as an AAV vector or a lentiviral vector.

[0219] The kit may further include a gRNA specific to the TRAC gene for inserting the CAR coding sequence into the TRAC gene. In other examples, the kit may further include a gRNA specific to the β2M gene for inserting the CAR coding sequence into the β2M gene. In other examples, the kit may further include a gRNA specific to the TGFBRII gene for inserting the CAR coding sequence into the TGFBRII gene. In other examples, the kit may further include a gRNA specific to the Reg-1 gene for inserting the CAR coding sequence into the Reg-1 gene. In other examples, the kit may further include a gRNA specific to the cbl-b gene for inserting the CAR coding sequence into the cbl-b gene.

[0220] In other embodiments, the kits disclosed herein may comprise a therapeutic T cell population as disclosed for the intended therapeutic purpose.

[0221] Any kit disclosed herein may further include instructions for preparing therapeutic T cells or therapeutic applications of therapeutic T cells. In some instances, the included instructions may include a description of genetically engineering one or more target genes disclosed herein using gene editing components. In other instances, the included instructions may include a description of how to introduce a nucleic acid encoding a CAR construct into a T cell to prepare a therapeutic T cell.

[0222] Alternatively, the kit may further include instructions for administering the therapeutic T cells disclosed herein to achieve the desired activity, such as eliminating the disease cells targeted by the CAR expressed on the therapeutic T cells. The kit may further include a description of selecting a subject suitable for treatment based on identifying whether the subject needs treatment. Instructions related to the use of therapeutic T cells described herein generally include information on the dosage, dosing regimen, and route of administration for the intended treatment. The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. The instructions provided in the kit disclosed herein are generally written instructions on a label or package insert. The label or package insert indicates that the therapeutic T cells are used to treat, delay the onset, and / or alleviate a disease or disorder in a subject.

[0223] The kit provided herein adopts suitable packaging. Suitable packaging includes but is not limited to vials, bottles, jars, soft packaging, etc. It is also considered that the packaging used in combination with a specific device, such as an infusion device for administering therapeutic T cells. The kit can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). The container can also have a sterile access port.

[0224] The kit may optionally provide additional components, such as buffers and interpretative information. Typically, the kit comprises a container and a label or package insert located on or associated with the container. In some embodiments, the disclosure provides an article comprising the contents of the above-described kit.

[0225] Sequence Listing

[0226] Table 1. Anti-GPC3 CAR components and their sequences

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242] Table 2. sgRNA sequences and target sequences

[0243]

[0244]

[0245]

[0246] *: 2'-O-methyl phosphorothioate residue

[0247] "n" refers to the spacer sequence at the 5' end

[0248] Table 3. Exemplary donor template sequences

[0249]

[0250]

[0251]

[0252] General techniques

[0253] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. These techniques are fully explained in the literature, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1989) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths and DG Newell, eds. August 1993) J. Wiley and Sons; Methods in Enzymology (Academic Press); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells Cells (JM Miller and MP Calos, 1987); Current Protocols in Molecular Biology (FM Ausubel et al., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JE Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA 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: an academic manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Press, 1995); DNA Cloning: A practical Approach, Volumes I and II (DN Glover, ed., 1985); Nucleic Acid Hybridization (BD Hames & S. J. Higgins eds. (1985); Transcription and Translation (BD Hames & S. J. Higgins, eds., (1984); Animal Cell Culture (RI Freshney, ed. (1986); Immobilized Cells and Enzymes (lRL Press, (1986); and B. Perbal, A practical Guide To Molecular Cloning (1984); FM Ausubel et al. (eds.).

[0254] It is believed that those skilled in the art can make the most of the present invention based on the above description without further detailed description. Therefore, the following specific embodiments should be interpreted as merely illustrative and not limiting the remainder of the present disclosure in any way. For the purpose or subject matter cited herein, all publications cited herein are incorporated herein by reference. Example

[0255] Certain aspects of the above-described embodiments are disclosed in further detail in the following examples, which are not intended in any way to limit the scope of the present disclosure.

[0256] Example 1. TRAC - / B2M - / Generation and characterization of anti-Glypican3 (GPC3) CAR+T cells

[0257] This example describes the generation and characterization of allogeneic human T cells that lack expression of the TRAC gene and the β2M gene and express a chimeric antigen receptor (CAR) targeting Glypican 3 (GPC3) (anti-GPC3 CAR).

[0258] Generation of anti-GPC3 CAR-T cells

[0259] Six unique exemplary GPC3 binders were generated, with either a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain, as shown in Table 4.

[0260] Table 4. Allogeneic CAR-T cells with GPC3 constructs

[0261] cell GPC3 conjugate Costimulatory domain SEQ ID ID 1524 HN3 single domain CD28 10 1524b HN3 single domain 4-1BB 13 1525 HkDJ CD28 25 1525b HkDJ 4-1BB 28 1526 HkDJ CD28 31 1526b HkDJ 4-1BB 34

[0262] Briefly, PBMCs were thawed and treated with TransAct TM Activation. After 0-3 days, cells were electroporated with Cas9:sgRNA RNP complexes and transduced with adeno-associated adenoviral vector (AAV) to generate genetically engineered TRAC - / B2M - / Anti-GPC3 CAR + T cells, wherein a nucleic acid encoding an anti-GPC3 CAR is inserted at the TRAC locus.

[0263] The sgRNA that forms RNP with the Cas9 enzyme is introduced into T cells by single or multiple electroporation events. After electroporation, the cells are transduced with recombinant AAV to introduce a donor template encoding anti-GPC3 CAR. Recombinant AAV serotype 6 (AAV6) containing one of the nucleotide sequences encoding the anti-GPC3 CAR listed in Table 1 above is delivered to activated human T cells together with Cas9: sgRNA RNP (1 μM Cas9, 5 μM GRNA). The following sgRNAs are used: TRAC (SEQ ID NO: 38) and β2M (SEQ ID NO: 42). In general, unmodified or modified versions of sgRNA can be used. Exemplary gRNA sequences are shown in Table 2.

[0264] CAR expression and editing efficiency assessment

[0265] CAR expression was assessed by flow cytometry using biotinylated GPC3 antigen (1 μg) followed by incubation with APC-conjugated streptavidin (200 ng). Figure 1A As shown, CAR expression was similar in all anti-GPC3 CAR constructs.

[0266] The editing efficiency of TRAC and β2M knockout was assessed by flow cytometry and TIDE analysis. Figure 1B As shown, similar levels of editing efficiency of TRAC and β2M genes were observed in CAR-T cells expressing the above anti-GPC3 CAR constructs.

[0267] Assessment of CD4:CD8 T cell ratio

[0268] The frequencies of CD4 and CD8 T cells in these cell cohorts and the differential profiles of CAR T cells were also determined by flow cytometry. The average frequencies were as follows Figure 2A and 2B There were no significant changes in the CD4+ to CD8+ cell ratios in any of the CAR T cell populations. Figure 2A All cell populations were highly enriched for central memory cells. Figure 2B .

[0269] Evaluation of immune checkpoint molecules

[0270] The expression of two immune checkpoint molecules, PD1 and Lag3, was evaluated in the cell population. PD1, as an immune checkpoint molecule, downregulates T cell activity during immune responses to prevent autoimmune tissue damage (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.

[0271] like Figure 3 As shown in Table 5, no significant changes in PD1 and LAG-3 levels were observed in any cell population.

[0272] Table 5. PD1 and LAG3 expression in CAR-T cells expressing anti-GPC3 CAR

[0273] cell Lag-3+(%) PD1+(%) 1524 15 8 1524b 14 5 1525 16 7 1525b 13 3 1526 14 5 1526b 13 4

[0274] Example 2. In vitro and in vivo cytotoxicity of anti-GPC3 CART cells

[0275] This example shows that the anti-GPC3 CAR T cells described in Example 1 selectively cleave GPC3 in vitro and in vivo. + The ability of cancer cells.

[0276] In vitro cytotoxicity

[0277] Anti-GPC3 CAR T cells were plated with HepG2 target cells with high GPC3 expression, A498 cells that do not express GPC3, or Huh7 cells that express low levels of GPC3 at different ratios. One day later, the number of live target cells and T cells was counted. Figures 4A-4CAs shown, CAR T cells specifically kill target cells expressing GPC3 antigen (HepG2 and Huh7 cells), but not GPC3-negative target cells (A498 cells). Compared with target cells expressing low levels of GPC3 (such as Huh7 target cells), high cytotoxicity was observed when target cells expressed high GPC3 levels (such as HepG2 target cells).

[0278] In vivo cytotoxicity

[0279] The in vivo efficacy of anti-GPC3 CAR T cells in reducing tumor growth was examined using a mouse xenograft model.

[0280] Female NSG mice were implanted subcutaneously in the right flank with 50% / 50% HepG2 tumor cells in culture medium (5x10 6 When tumors were clearly visible, mice were randomly divided into 5 groups and injected intravenously with CAR T cells (6x10 per mouse). 6 CAR + Tumor volume was assessed every few days and displayed on Figure 5 and Table 6. CAR-T cells expressing 1524 and 1525 anti-GPC3 CARs showed the highest anti-tumor activity compared to other anti-GPC3 constructs. N / A indicates that there were no surviving mice for measuring tumor volume.

[0281] Table 6. Tumor volume

[0282]

[0283]

[0284] N / A = No surviving animals were used for measurement

[0285] Example 3. Production and characterization of CAR T cells with gene editing for added potency

[0286] This example describes allogeneic human TRAC - / B2M - / Anti-GPC3 CAR + Generation and characterization of T cells that were also edited for TGFBRII, Regnase-1, and CBLB genes as listed in Table 7.

[0287] Table 7. Genetically engineered CAR-T cells with GPC3 constructs

[0288]

[0289] Generate TRAC - / B2M - / anti-GPC3CART cells with additional disruption of TGFbRII / Reg-1 or TGFbRII / cbl-b

[0290] In brief, 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 and CBLB or Regnase-1 (REG-1) knockouts. The sgRNA that forms RNPs with the Cas9 enzyme is introduced into T cells by single or multiple electroporation events. After electroporation, cells are transduced with recombinant AAV to introduce donor templates encoding anti-GPC3 CARs (see Example 1 and Table 1). Recombinant AAV serotype 6 (AAV6) containing one of the nucleotide sequences encoding one of the anti-GPC3 CARs is delivered to activated human T cells together with Cas9:sgRNA RNPs (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 sgRNAs may also be used. Exemplary gRNA sequences are shown in Table 2.

[0291] Assessment of T cell expansion

[0292] Engineered T cells were counted periodically to assess T cell expansion. Figure 6 As shown in Table 8, knocking out the TGFBRII gene and the Regnase-1 gene or the CBLB gene had no effect on cell growth, because the additionally edited cells were expressed as TRAC - / B2M - / Anti-GPC3 CAR + The cells proliferated at similar rates.

[0293] Table 8. T cell expansion

[0294]

[0295] CAR expression and editing efficiency assessment

[0296] CAR expression was assessed by flow cytometry using biotinylated GPC3 antigen (1 μg) followed by incubation with APC-conjugated streptavidin (200 ng). CAR expression was similar in all anti-GPC3 CAR constructs. Figure 7 .

[0297] The editing efficiency of TRAC, β2M, TGFBRII, and Regnase-1 knockdown was assessed by flow cytometry and TIDE analysis. It was observed that TRAC and β2M had similar editing efficiency levels in the presence or absence of Regnase-1 and TGFBRII. Figures 8A-8C

[0298] CBLB KO was assessed by western blot analysis, indicating that CBLB was efficiently knocked out in all anti-GPC3 CART cells examined. Fig. 9 .

[0299] Assessment of CD4:CD8 T cell ratio

[0300] The frequencies of CD4 and CD8 T cells in these cell cohorts were also determined by flow cytometry. Fig.10 Disruption of the Regnase-1 and TGFBRII genes did not show a significant effect on the ratio of CD4+ to CD8+ cells in the CAR-T cell population.

[0301] In summary, the results provided in this example show that in anti-GPC3 CAR-T cells with disrupted TRAC and B2M, genetic disruption of the TGFbRII gene alone together with disruption of the Reg1 gene or the cbl-b gene did not show a significant effect on the CAR-T cell characteristics.

[0302] Example 4. In vitro cytotoxicity of anti-GPC3 CAR T cells with potent gene editing

[0303] This example demonstrates the ability of the anti-GPC3 CAR T cells described in Example 3 above to selectively lyse GPC3+ cancer cells.

[0304] Study 1

[0305] Anti-GPC3 CAR T cells were plated with HepG2 target cells with high GPC3 expression or with A498 cells that do not express GPC3 at different ratios. One day later, the number of live target cells and T cells were counted. Figures 11A-11B As shown in Tables 9-10 below.

[0306] Cytotoxicity measurements were normalized as follows to account for the loss of target cells during washing after co-culture:

[0307] Cytotoxicity CAR-Conc(A) =100*(1-(luminescence CAR-Conc(A) / Luminous AAV-Conc(A) ),

[0308] Wherein cytotoxicity is the percentage measurement of the inverse of the luminescence of "CAR-Conc (A)" divided by the luminescence of "AAV-Conc (A)", "CAR-Conc (A)" refers to a given ratio of T cells to target cells in a CAR T cell population, and "AAV-Conc (A)" refers to a given ratio of T cells to target cells in a control (AAV-) population. When activated by GPC3+ target cells, increased cell proliferation was also observed. Cytotoxicity against GPC3- target cells was not observed, as shown in Table 10 below.

[0309] Table 9. Cytotoxicity of HepG2 (GPC3 高 )(%)

[0310]

[0311] Table 10. Cytotoxicity of A498 (GPC3 - )(%)

[0312]

[0313] Cytokine secretion

[0314] Cytokine secretion by these CAR T cells in the presence of GPC3-positive target cells was also measured. Figures 12A-12B As shown in Tables 11-12, the secretion of IFN-γ and granzyme A showed a ratio-dependent increase.

[0315] Table 11. IFN-γ secretion (pg / ml)

[0316]

[0317] Table 12. Granzyme A (pg / ml)

[0318]

[0319] Study 2

[0320] In vitro growth curves of cells with or without potency editing were measured at different days after electroporation. The data are shown in Table 13 and demonstrate that the cells are in good condition.

[0321] Table 13. Cell fold expansion

[0322]

[0323]

[0324] Compared with 1524 anti-GPC3 CAR T cells, potency-edited cells showed lower CAR knock-in and B2M knock-out and had similar TRAC - The ratio of CD4 / CD8 cells in the cell population was similar under all conditions.

[0325] HepG2 target cells with high surface expression of GPC3 were used to measure the specific cytotoxicity of these cells to cells expressing GPC3. A498 cells that do not express GPC3 were used as negative controls. The cytotoxicity was measured relative to AAV cells using chemiluminescence and presented in Tables 14-15. All cells showed good anti-GPC3 specific cytotoxicity. "E:T ratio" represents the ratio of effector cells to target cells. "0" means that no cytotoxicity was observed.

[0326] Table 14. Cytotoxicity of HepG2 cells (high GPC3 expression) (%)

[0327]

[0328] Table 15. Cytotoxicity of A498 cells (GPC3 negative) (%)

[0329]

[0330] Thus, all engineered cells showed good proliferation capacity, specific cytotoxicity against the target, and were considered phenotypically normal compared with equivalent CAR T cells used in in vivo studies.

[0331] Example 5. In vitro cytotoxicity of anti-GPC3 CART cells with potent gene editing The engineered CAR T cells provided in the previous examples were tested in a mouse model of liver cancer.

[0332] In short, 10x10 6 Hep3B cancer cells were injected into the left flank of mice (5 per group) and allowed to grow to approximately 125 mm 3 Once the tumor reached the target volume, 8x10 6 CAR T cells. 30 days after the administration of CAR T cells (54 days after the start of the study), the mice were once again challenged with another tumor cell in the right flank. Any mice showing high tumor burden were removed and euthanized. Groups with reduced numbers of mice are indicated by "*". Tumor volumes of primary tumors were measured regularly and are shown in Table 16. The data show that 1524 is more efficient than untreated controls for primary tumors. The addition of efficacy editing, TGFBRII, Regnase-1, or CBLB greatly improved primary tumor control of 1524CAR T cells.

[0333] Table 16. Primary tumor volume (mm 3 )

[0334]

[0335]

[0336] The tumor volumes of the secondary tumors administered as a secondary challenge are presented in Table 17. In this case, a new group of mice was utilized as untreated controls, since all untreated mice challenged with the primary tumor were euthanized.

[0337] Table 17. Secondary (re-challenge) tumor volume (mm 3 )

[0338]

[0339] The body weight of mice was monitored throughout the study. Data are shown in Table 18 as a measure of the change in body weight since the start of the study.

[0340] Table 18. Average body weight change (%)

[0341]

[0342]

[0343] Example 6. Generation and characterization of anti-GPC3 CAR-T cells for autologous cell therapy

[0344] This example describes the construction and characterization of anti-GPC3 CAR-T cells for autologous cell therapy.

[0345] A lentiviral vector was constructed for delivering the coding sequence of the anti-GPC3 CAR (see Example 1 and Table 1) to immune cells to express the anti-GPC3 CAR. Briefly, a pCCL-c-MNDU3-X2 plasmid carrying a mutated WPRE (mWPRE) and the coding sequence of the anti-GPC3 CAR (as a gene of interest or GOI) was constructed. The general structure of the construct is shown below;

[0346] 5'LTR-Gag'-RRE-cPPT-(Promoter)-GOI-mWPRE-3-LTR

[0347] This plasmid carries the Moloney murine leukemia virus (MMLV) LTR (MNDU3) promoter, which is known to be suitable for T cells and other leukocytes. The mWPRE can be obtained from known plasmids carrying mWPRE (e.g., pENTR-L5-WPRE-L2, Addgene), or by mutagenesis of wild-type WPRE.

[0348] First, the desired cassette with "anti-GPC3 CAR-mWPRE" or "MNDU3 promoter-anti-GPC3 CAR-mWPRE" is constructed on the intermediate plasmid. In short, after the cassette is PCR amplified and DNA connected to the intermediate plasmid, the ligation mixture is used to transform E. coli cells. The generated clones are screened to select clones with plasmids with the desired cassette. The cassette is excised and cloned into the pCCL-c-MNDU3-X2 plasmid. After sequencing to verify that the desired cassette has been inserted, the resulting plasmid is used to transduce packaging cells, together with plasmids capable of producing proteins required for lentiviral packaging (e.g., gag / pol, Rev, and VSV-G) to produce lentiviral particles, which are then harvested.

[0349] Peripheral blood lymphocytes (PBL) were obtained from cells that needed to eliminate GPC3 + The cells are electroporated using Cas9:sgRNA RNP complexes to genetically edit TGFbRII, Reg-1, and / or CBLB, and transduced with the above-mentioned lentivirus, which carries nucleic acid encoding the anti-GPC3 CAR construct. In some cases, transduction is performed in the presence of RetroNectin to improve transduction efficiency. The sgRNA sequences for genetic editing of TGFbRII, REG-1, and CBLB genes are shown in Table 2. The resulting anti-GPC3 CAR-T cells with one or more of the disrupted TGFbRII, REG-1, and CBLB genes are collected.

[0350] The ability of these engineered anti-GPC3CAR-T cells to kill GPC3+ target cells was tested using in vitro proliferation, cytotoxicity, and cytokine release assays; and in vivo cytotoxicity, persistence, and cytokine analysis using the NSG mouse model according to the guidance provided in Examples 2-4 above. Using flow cytometry, cells recovered from mouse blood, spleen, and other tissues were used to determine the persistence, biodistribution, and phenotype of engineered anti-GPC3 CAR cells.

[0351] Example 7. Autologous Cell Therapy Using Anti-GPC3 CAR-T Cells

[0352] This example describes autologous cell therapy for patients with hepatocellular carcinoma (HCC) using anti-GPC3 CAR-T cells prepared from peripheral blood lymphocytes (PBL) obtained from the patients.

[0353] PBLs were obtained from patients with HCC and genetically engineered according to the instructions provided in Example 6 to generate engineered T cells expressing anti-GPC3 CAR and having disrupted one or more of the TGFbRII, REG-1, and CBLB genes.

[0354] The engineered anti-GCP3 CAR-T cells thus generated were administered to patients. The ability of anti-GPC3CART cells to reduce tumor burden, persist in vivo, and differentiate in vivo was detected according to routine practice. Cells recovered from the blood were used to determine the persistence, biodistribution, and phenotype of engineered anti-GPC3 CAR cells using flow cytometry.

[0355] Other Implementations

[0356] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is only a general example of a series of equivalent or similar features.

[0357] From the above description, those skilled in the art can easily determine the essential characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, other embodiments are also within the claims.

[0358] Equivalent

[0359] Although several inventive embodiments of the present disclosure have been described and illustrated herein, a person of ordinary skill in the art will readily conceive of a variety of other means and / or structures for performing functions and / or obtaining results and / or one or more advantages described herein, and each such variation and / or modification is considered to be within the scope of the inventive embodiments described herein. More generally, it will be readily understood by those skilled in the art that all parameters, dimensions, materials, and configurations described herein are exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on the specific application or application in which the teachings of the present invention are used. Those skilled in the art will recognize or be able to determine many equivalents of the specific inventive embodiments described herein using only routine experiments. Therefore, it should be understood that the foregoing embodiments are presented only by way of example, and within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced in a manner different from that specifically described and claimed. Inventive embodiments of the present disclosure relate to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually contradictory, is included within the scope of the invention disclosed herein.

[0360] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0361] All references, patents, and patent applications disclosed herein are hereby incorporated by reference for the subject matter set forth in each document, which in some cases may be incorporated in its entirety.

[0362] The indefinite articles "a" and "an" as used in the specification and claims should be understood to mean "at least one", unless explicitly stated to the contrary.

[0363] The phrase "and / or" used in the specification and claims should be understood to mean "one or both" of the elements so combined, i.e., elements that exist jointly in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" elements so connected. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprising", a reference to "A and / or B" may refer, in one embodiment, to only B (optionally including elements other than A); in another embodiment, to both A and B (optionally including other elements); etc.

[0364] As used herein in the specification and claims, "or" shall 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" shall be interpreted as inclusive, i.e., including at least one of a plurality of elements or a list of elements, but also including a plurality of elements, and optionally other unlisted items. Only terms that clearly indicate the opposite, such as "only one" or "exactly one", or when used in the claims, "consisting of...", will refer to only one element in a plurality of elements or a list of elements. In general, the term "or" as used herein shall be interpreted as indicating an exclusive alternative (i.e., "either one or the other, but not both") only when it is preceded by an exclusive term such as "either", "one of", "only one of", or "exactly one of". When used in the claims, "consisting essentially of..." shall have its ordinary meaning used in the field of patent law.

[0365] As used herein, the term "approximately" means within an acceptable error range for a particular value determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within an 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 still more preferably up to ±1% of a given value. When describing a particular value in the present application and claims, unless otherwise stated, the term "about" is implicit and means in this article to be within an acceptable error range for the particular value.

[0366] As used herein in the specification and claims, the phrase "at least one" referring to a list of one or more elements should be understood to refer to at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements referred to by the phrase "at least one" may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a 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") may, in one embodiment, refer to at least one, optionally including more than one, A, without B (and optionally including elements other than B); in another embodiment, refer to at least one, optionally including more than one, B, without A (and optionally including elements other than A); in yet another embodiment, refer to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0367] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order of the steps or actions of the method as described unless explicitly stated to the contrary.

Claims

1. A genetically engineered T cell population, 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 comprising: (i) a disrupted T cell receptor alpha chain constant region (TRAC) gene, (ii) a disrupted beta-2 microglobulin (β2M) gene, (iii) disrupted transforming growth factor beta receptor II (TGFbRII) gene, (iv) disrupted Regnase-1 (Reg1) gene, (v) a disrupted Casitas B lineage lymphoma oncogene-B (CBLB) gene, or (vi) A combination of any one of (i) to (v).

2. The genetically engineered T cell population of claim 1, wherein the anti-GPC3 CAR comprises: (a) Binds to the extracellular domain of GPC3; (b) a transmembrane domain; and (c) An intracellular domain comprising (i) a co-stimulatory signaling domain and (ii) a CD3ζ cytoplasmic signaling domain. 3 . The genetically engineered T cell population according to claim 2 , wherein the extracellular domain 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). 4 . The genetically engineered T cell population of claim 3 , wherein the extracellular domain comprises the anti-GPC3 VHH comprising complementarity determining regions (CDRs) identical to those in SEQ ID NO:

9. The genetically engineered T cell population of claim 3 , wherein the anti-GPC3 VHH comprises the amino acid sequence of SEQ ID NO:

9.

6. The genetically engineered T cell population according to claim 3, wherein the extracellular domain comprises the anti-GPC3 scFv, which comprises a heavy chain variable (V H ) region and light chain variable (V L ) region, the heavy chain variable (V H ) region comprises CDRs identical to those in SEQ ID NO: 22, the light chain variable (V L ) region is a CDR identical to the CDR in SEQ ID NO:

21.

7. The genetically engineered T cell population of claim 6, wherein the anti-GPC3 scFv comprises a V sequence as shown in SEQ ID NO:

22. H and V as shown in SEQ ID NO: 21 L。 8. The genetically engineered T cell population of claim 7, wherein the anti-GPC3 scFv comprises the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO:

24.

9. The genetically engineered T cell population of any one of claims 2-8, wherein the co-stimulatory domain is a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain.

10. The genetically engineered T cell population according to any one of claims 2-9, wherein the transmembrane domain is a CD8 transmembrane domain.

11. The genetically engineered T cell population of claim 2, wherein the anti-GPC3 CAR comprises the amino acid sequence of SEQ ID NO: 11, 14, 26, 29, 32 or 35.

12. The genetically engineered T cell population of any one of claims 1-11, wherein the one or more disrupted genes of (b) comprise the disrupted TRAC gene and the disrupted β2M gene.

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

14. The population of genetically engineered T cells of claim 12, wherein the one or more disrupted genes of (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.

15. The genetically engineered T cell population of any one of claims 1-11, wherein the one or more disrupted genes of (b) comprise the disrupted TGFBRII gene, the disrupted Reg-1 gene, the disrupted CBLB gene, or a combination thereof. The genetically engineered T cell population according to claim 15 , comprising a wild-type TRAC gene, a wild-type β2M gene or a combination thereof.

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

18. The population of genetically engineered T cells of claim 17, wherein the endogenous genetic locus is located within the disrupted TRAC gene, the disrupted β2M gene, the disrupted TGFbRII gene, the disrupted Reg-1 gene, or the disrupted CBLB gene.

19. The population of genetically engineered T cells of claim 18, wherein the endogenous genetic locus is located within the disrupted TRAC gene.

20. The genetically engineered T cell population of claim 19, wherein the disrupted TRAC gene comprises a deletion of SEQ ID NO: 58, which is optionally replaced by the nucleotide sequence encoding the anti-GPC3 CAR.

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

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

23. The method of claim 22, wherein step (a) comprises delivering the TRAC gRNA and the β2M gRNA to the T cell population.

24. The method of claim 23, wherein step (a) further comprises delivering TGFBRII gRNA, Reg1 guide, CBLB guide, or a combination thereof to the T cell population.

25. The method of claim 23, wherein step (a) further comprises delivering to the T cell population (i) TGFBRII gRNA and Reg1 guide, or (ii) TGFBRII guide and CBLB guide.

26. The method of claim 22, wherein step (a) comprises delivering TGFBRII gRNA, Reg1 guide, CBLB guide, or a combination thereof to the T cell population.

27. The method according to any one of claims 22 to 26, wherein: (i) the TRAC guide is specific to a TRAC gene target sequence comprising a nucleotide sequence of SEQ ID NO:58; (ii) the β2M guide is specific to a β2M gene target sequence comprising a 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.

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

55.

29. The method of any one of claims 22-28, 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 of any one of claims 22-29, wherein the TRAC guide, the β2M guide, the TGFBRII guide, the Reg1 guide and / or the CBLB guide comprises one or more modifications.

31. A method according to claim 29 or claim 30, wherein: (i) the TRAC guide comprises the nucleotide sequence of SEQ ID NO:37 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.

32. The method of any one of claims 22-31, wherein the one or more RNA-guided nucleases comprises a Cas9 nuclease, which is optionally a Streptococcus pyogenes Cas9 nuclease.

33. The method of any one of claims 22-32, wherein the vector in (a)(iii) comprises a donor template, wherein the nucleic acid encoding the anti-GPC3 CAR is flanked by an upstream segment and a downstream segment, and wherein the upstream segment and the downstream segment are homologous to the endogenous genetic locus of the T cell, allowing the nucleic acid encoding the anti-GPC3 CAR to be inserted into the endogenous genetic locus.

34. The method of claim 33, wherein the endogenous genetic locus is located within the disrupted TRAC gene, the disrupted β2M gene, the disrupted TGFbRII gene, the disrupted Reg-1 gene, or the disrupted CBLB gene.

35. The method of claim 33, wherein the endogenous genetic locus is located within the disrupted TRAC gene.

36. The method according to claim 35, wherein the upstream fragment is shown as SEQ ID NO:71, and / or wherein the downstream fragment is shown as SEQ ID NO:

74.

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

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

39. The method of any one of claims 22-38, wherein the T cell population comprises human T cells, which are optionally human primary T cells.

40. The method of any one of claims 22-39, wherein the T cell population is obtained from one or more healthy human donors.

41. The method of any one of claims 22-39, wherein the T cell population is obtained from a human patient suffering from a GPC3+ cancer.

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

43. The method of claim 42, wherein the subject is a human patient suffering from a GPC3+ cancer.

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

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

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

47. The method of claim 46, wherein the genetically engineered T cell population is as described in any one of claims 15-16.

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

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

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

51. The anti-GPC3 CAR of 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 of any one of claims 49-51.

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

54. The nucleic acid of claim 53, wherein the vector is a viral vector, which is optionally an AAV vector or a lentiviral vector.

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