Production of immune cells

Abstract

The present invention relates to methods for producing immune cells expressing therapeutic antigen receptors. An immune cell is provided, the immune cell comprising a heterologous expression cassette. The heterologous expression cassette comprises (a) a coding sequence for generating a TCR, (b) a constitutive promoter operably linked to the coding sequence, (c) a 5 '-targeting site, and optionally (d) a 3'-targeting site. An expression construct is then introduced into the immune cell at the site of the heterologous expression cassette. The expression construct comprises a coding sequence for a therapeutic antigen receptor, and then the therapeutic antigen receptor is expressed in the immune cell. Methods, reagents for use in the methods, and immune cells produced by the methods are provided.

Description

Technical Field

[0001] The present invention relates to the generation of immune cells, for example for use in immunotherapy. Background Art

[0002] Immunotherapy is expected to change the landscape of cancer treatment and to achieve long-term survival (McDermott et al., Cancer Treat Rev. 2014 Oct;40(9):1056-64). There is a clear unmet medical need for new immunomodulatory drugs that expand the range of eligible patient populations and the range of tumor types. In addition, new agents are needed to enhance the magnitude and duration of anti-tumor responses. The development of these agents has been made possible by a deeper understanding of the basic principles controlling T cell immunity over the past two decades (Sharma and Allison, Cell 2015 Apr 9;161(2):205-14). This generally requires tumor-specific immune cells, such as CD4+ and CD8+ T cells, to recognize tumor-associated peptide antigens presented by MHC molecules. In some cases, different vaccination strategies and adoptive transfer of ex vivo expanded tumor-infiltrating lymphocytes have demonstrated the ability of tumor-specific immune cells to treat advanced cancers (Rosenberg et al., Nat Med 2004 Sep;10(9):909-15).

[0003] However, current adoptive immune cell therapies are limited by the lack of suitable patient- and tumor-specific immune cells, and therapeutically sufficient and functional antigen-specific immune cells are required for effective immunotherapy. Summary of the invention

[0004] The present inventors have developed methods involving the generation of immune cells comprising a heterologous expression cassette for "placeholder" production of a T cell receptor (TCR). After generation, the immune cells can then be sensitized for therapeutic use in a patient by replacing the heterologous expression cassette with an expression construct encoding a therapeutic antigen receptor, such as an antigen receptor that binds to the patient's cancer cells. These methods can be used, for example, to generate immune cells for immunotherapy, such as allogeneic immune cells, specifically to generate "personalized" immune cells with therapeutic antigen receptors that are selected for binding to the patient's cancer cells.

[0005] A first aspect of the present invention provides a method for producing immune cells expressing a therapeutic antigen receptor, the method comprising:

[0006] (i) providing an immune cell comprising a heterologous expression cassette,

[0007] Wherein the heterologous expression cassette comprises:

[0008] (a) a coding sequence for producing a T cell receptor (TCR),

[0009] (b) a constitutive promoter operably linked to the coding sequence,

[0010] (c) target site, and

[0011] (ii) introducing an expression construct comprising a coding sequence for a therapeutic antigen receptor into said immune cell at the site of said heterologous expression cassette,

[0012] The therapeutic antigen receptor is allowed to be expressed in the immune cell.

[0013] In some embodiments of the first aspect of the invention, the heterologous expression cassette can be replaced by the expression construct comprising the coding sequence for the therapeutic antigen receptor. For example, a method for generating an immune cell expressing a therapeutic antigen receptor can comprise:

[0014] (i) providing an immune cell comprising a heterologous expression cassette,

[0015] Wherein the heterologous expression cassette comprises:

[0016] (a) a coding sequence for producing a T cell receptor (TCR),

[0017] (b) a constitutive promoter operably linked to the coding sequence, and

[0018] (c) target site, and

[0019] (ii) replacing said heterologous expression cassette in said immune cell with an expression construct comprising a coding sequence for a therapeutic antigen receptor,

[0020] The therapeutic antigen receptor is allowed to be expressed in the immune cell.

[0021] A second aspect of the present invention provides a method for producing immune cells expressing a therapeutic antigen receptor, the method comprising:

[0022] (i) providing an induced pluripotent stem cell (iPSC), said iPSC comprising a heterologous expression cassette at a site in the genome of said iPSC,

[0023] Wherein the heterologous expression cassette comprises:

[0024] (a) a coding sequence for producing a T cell receptor (TCR),

[0025] (b) a constitutive promoter operably linked to the coding sequence, and

[0026] (c) target site,

[0027] (ii) differentiating the iPSCs into immune cells, and

[0028] (iii) introducing an expression construct comprising a coding sequence for a therapeutic antigen receptor into said immune cell at the site of said heterologous expression cassette,

[0029] The therapeutic antigen is allowed to be expressed in the immune cells.

[0030] For example, a method for generating immune cells expressing a therapeutic antigen receptor may comprise:

[0031] (i) providing an induced pluripotent stem cell (iPSC), said iPSC comprising a heterologous expression cassette at a site in the genome of said iPSC,

[0032] Wherein the heterologous expression cassette comprises:

[0033] (a) a coding sequence for producing a T cell receptor (TCR),

[0034] (b) a constitutive promoter operably linked to the coding sequence, and

[0035] (c) target site,

[0036] (ii) differentiating the iPSCs into immune cells, and

[0037] (iii) replacing said heterologous expression cassette in said immune cell with an expression construct comprising a coding sequence for a therapeutic antigen receptor,

[0038] causing said coding sequence for said therapeutic antigen to be expressed in said immune cell,

[0039] This generates immune cells that express receptors for the therapeutic antigen.

[0040] iPSCs may be provided in the method according to the second aspect by transfecting IPSCs with a nucleic acid comprising the heterologous expression cassette, such that the heterologous expression cassette is integrated into the genome of the IPSCs.

[0041] A third aspect of the present invention provides an immune cell comprising a heterologous expression cassette integrated into its genome,

[0042] Wherein the heterologous expression cassette comprises:

[0043] (a) a coding sequence for producing a T cell receptor (TCR),

[0044] (b) a constitutive promoter operably linked to the coding sequence, and

[0045] (c) Targeting site.

[0046] In some embodiments of the first to third aspects, the therapeutic antigen receptor can specifically bind to cancer cells.

[0047] A fourth aspect of the present invention provides an IPSC comprising a heterologous expression cassette integrated into its genome,

[0048] Wherein the heterologous expression cassette comprises:

[0049] (a) a coding sequence for producing a T cell receptor (TCR),

[0050] (b) a constitutive promoter operably linked to the coding sequence, and

[0051] (c) Targeting site.

[0052] In some embodiments of the first to fourth aspects, the targeting site may be a 5' targeting site. The heterologous expression cassette may further comprise a 3' targeting site.

[0053] The heterologous expression cassette according to the first to fourth aspects may further comprise a coding sequence for a poly(A) sequence.

[0054] The fifth aspect of the present invention provides an immune cell population produced by the method according to the first aspect or the second aspect.

[0055] The sixth aspect of the present invention provides a pharmaceutical composition, which comprises the immune cell population according to the fifth aspect and a pharmaceutically acceptable excipient.

[0056] The seventh aspect of the present invention provides a treatment method, which comprises administering a therapeutically effective dose of the immune cell population according to the fifth aspect to an individual in need thereof.

[0057] The individual may be suffering from a cancer condition.

[0058] Other aspects and embodiments of the invention are described in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Schematic diagram showing an example of a six-stage method for generating T cells from iPSCs.

[0060] In stage 4 in 15F2_AAVS1 - / A2M10LP and 16D5_AAVS1 A2M10LP / A2M10LP The exchange of the placeholder A2M10 to A2M4 was performed in early iT cell progenitors. The placeholder exchange was performed on late iT cell progenitors at stage 6 after activation with CD3 / 28.

[0061] Figure 2 A schematic diagram of an example of a TCR "landing pad" is shown. A 'placeholder' TCR is integrated into the genome using CRISPR+AAV. Under the control of a constitutive promoter, this expression cassette drives the expression of the TCR required to promote full iT cell differentiation. The candidate therapeutic TCR replaces the 'placeholder' TCR during the final editing step of the iT-cell. CRISPR / Cas9 targeting of TRAC results in two (2) simultaneous events: (i) excision of the 'placeholder' TCR and replacement with a functional candidate TCR that will be under the control of an endogenous promoter, and (ii) knockout of the natural endogenous TRAC locus.

[0062] Figure 3 A targeting strategy for inserting a TCR "landing pad" into the last exon of a coding gene is described. The targeting vector contains left and right homology arms (HA) corresponding to 300-1000 nucleotides of the genomic DNA 5' and 3' of the CRISPR guide RNA, a truncated TRAC domain containing a 5' landing pad excision sequence, a poly A signal (PA), an exogenous promoter, a 2A "like" skipping sequence, a "placeholder" TCR, and an additional PA sequence. The TRAC domain "placeholder" TCR contains a landing pad 3' excision site. After the monoallelic gene is integrated into the desired genomic location, the second targeting vector is designed to integrate after the landing pad is excised using a guide RNA targeting the excision site. The secondary targeting vector contains left and right homology arms (HA), a 2A "like" skipping sequence, and a therapeutic TCR. The TCR alpha chain of the therapeutic TCR contains a truncated TRAC domain. When the therapeutic TCR is knocked into the landing pad, the full-length TRAC domain is reconstructed.

[0063] Figure 4An alternative targeting strategy of inserting a TCR "landing pad" into the last exon of a coding gene is described. In this scenario, 5' and 3' excision sequences correspond to the B2M region (Chr 15 44715435 to 44715475) containing a guide RNA recognition sequence. The excision sequence is flanked by a placeholder TCR. The flanking region can be modified to contain any guide sequence of interest. The targeting vector contains left and right homology arms (HA), poly A signals (PA), exogenous promoters, 2A "like" skipping sequences, "placeholder" TCR, and additional PA sequences corresponding to 300-1000 nucleotides of genomic DNA 5' and 3' from the integration site. After the monoallelic gene is integrated into the desired genomic position, the second targeting vector is designed to integrate after the guide RNA excision of the landing pad using the targeted excision site. The secondary targeting vector contains left and right homology arms (HA), 2A "like" skipping sequences, and therapeutic TCR.

[0064] Figure 5 The targeting strategy of inserting TCR "landing pad" into the genomic "safe harbor" locus is described. The targeting vector comprises left and right homology arms (HA) corresponding to 300-1000 nucleotides of 5' and 3' genomic DNA from the integration site, a truncated TRAC domain comprising a 5' landing pad excision sequence, a poly A signal (PA), an exogenous promoter, a 2A "like" skipping sequence, a "placeholder" TCR, and an additional PA sequence. The TRAC domain in the "placeholder" TCR contains a landing pad 3' excision site. After the monoallelic gene is integrated into the desired locus, the second targeting vector is designed to integrate after the guide RNA using the targeted excision site excises the landing pad. The secondary targeting vector contains left and right homology arms (HA), an exogenous promoter, a 2A "like" skipping sequence, and a therapeutic TCR. The TCR alpha chain of the therapeutic TCR contains a truncated TRAC domain. When the therapeutic TCR is knocked into the landing pad, the full-length TRAC domain is reconstructed.

[0065] Figure 6The targeting strategy of inserting TCR "landing pad" into the genome "safe harbor" locus is described. In this scenario, 5' and 3' excision sequences correspond to the B2M region (Chr 15 44715435 to 44715475) containing the guide RNA recognition sequence. The excision sequence is flanked by the placeholder TCR. The flanking region can be modified to contain any guide sequence of interest. The targeting vector contains left and right homology arms (HA) corresponding to 300-1000 nucleotides of genomic DNA 5' and 3' from the integration site, excision domain, poly A signal (PA), exogenous promoter, 2A "sample" skipping sequence, "placeholder" TCR and other PA sequences. After the monoallelic gene is integrated into the desired genomic position, the second targeting vector is designed to integrate after the guide RNA excision of the landing pad using the targeted excision site. The secondary targeting vector contains left and right homology arms (HA), 2A "sample" skipping sequence and therapeutic TCR.

[0066] Figure 7 An AAV targeting vector for inserting a TCR landing pad with TRAC guide RNA into PTPRC exon 33 is described. The left and right homology arms correspond to chromosome-198755130-198756201 and chromosome-198,756,132-198,757,230, respectively (Ensembl version 104 - May 20210). The guide RNA sequence within the LHA has been mutated to prevent cleavage of the repair template. MAGE-A10 TCR ADB796 is expressed by an exogenous promoter (EF1a short). The landing pad TCR cassette contains two guide RNA sequences derived from TRAC exon 1. The 5' site is a site contained within the truncated TRAC domain (Chr14: 22,547,508-22,547,560), and the 3' site is located within the TRAC domain of MAGE-A10 TCR ADB796. Editing with a single guide RNA allows excision of the landing pad TCR.

[0067] Figure 8An AAV targeting vector for inserting a TCR landing pad with TRAC guide RNA into the AAVS1 safe harbor site PPP1r12C intron 1 is described. The left and right homology arms correspond to chromosomes Chr 19: 55115776-55116775 and Chr 19: 55114775-55115775 (Ensembl 104 version - May 2021), respectively. MAGE-A10 TCR ADB796 is expressed by an exogenous promoter (EF1a short). The landing pad TCR cassette contains two guide RNA sequences derived from TRAC exon 1. The 5' site is the site contained within the truncated TRAC domain (Chr14: 22,547,508-22,547,560), and the 3' site is located within the TRAC domain of MAGE-A10 TCR ADB796. Editing with a single guide RNA allows excision of the landing pad TCR. In the alternative changes shown, the positions of the α and β chains can be reversed.

[0068] Fig. 9 A targeting vector for inserting a therapeutic TCR (MAGE-A4 / B2 ADB959) into a TCR landing pad within PTPRC exon 33 is described. The left homology arm targets Chr 1: 198,754,605-198,756,226 (Ensembl 104 version - May 2021) and the right homology arm targets the TCRαTRAC domain BGH polyA and genomic DNA corresponding to Chr 1: 198,756,132-198,757,230 (Ensembl 104 version - May 2021). The nucleotide sequence within the TRAC domain of ABD959 has been mutated to prevent cleavage by the guide RNA used to excise the placeholder TCR. The design of the targeting vector allows the insertion of a 2A-like skipping sequence-tagged TCR into a frame with PTPRC exon 33.

[0069] Fig.10 A targeting vector for inserting a therapeutic TCR (MAGE-A4 / B2 ADB959) into a TCR landing pad within PPP1r12C intron 1 is described. The left homology arm targets Chr 19: 55,115,701-55,117,349 (Ensembl 104 version - May 2021) and the right homology arm targets the TCRαTRAC domain BGH polyA and genomic DNA corresponding to Chr 19: 55,114,725-55,115,825 (Ensembl 104 version - May 2021). The nucleotide sequence within the TRAC domain of ABD959 has been mutated to prevent cleavage by the guide RNA used to excise the placeholder TCR. Expression of ADB959 is regulated by an exogenous promoter.

[0070] Fig.11 An overview of the landing pad strategy is shown. A placeholder TCR is knocked into iPSC cells to support differentiation into CD8+ T cells. Following differentiation, the iT cells are gene-edited to exchange the placeholder TCR for a patient-specific therapeutic TCR.

[0071] FIG. 12 illustrates the editing strategy of the present invention. Fig. 12A Shown is the knock-in of a placeholder TCR (A2M10) cassette into the PPP1R12C (AAVS1) locus using the rAAV repair template ADB00794_001 to generate a novel universal cell library. The B2M guide sequence for TCR excision is highlighted. The TCR α and TCR β chains are separated by the P2A skipping sequence. Fig. 12B Shown is the replacement of the placeholder TCR (A2M10) cassette with the exchange TCR (A2M4) cassette using the rAAV repair template ADB01032_026. The TCR alpha and TCR beta chains are separated by a P2A skipping sequence.

[0072] Fig.13 Shown is the expression of the A2M10 placeholder TCR in an early progenitor iT cell line derived from a monoallelic placeholder TCR knock-in iPSC clone (15F2_AAVS1 - / A2M10LP ) and biallelic placeholder TCR knock-in iPSC clones (16D5_AAVS1 A2M10PL / A2M10LP ) differentiated.

[0073] FIG. 14 shows early 15F2_AAVS1 after CRISPR-Cas9-based gene editing. - / A2M10LP Expression and exchange of the A2M10 placeholder TCR in progenitor iT cells for the A2M4 TCR. A) Cells were mock electroporated and transduced with the A2M4 rAAV repair template at a multiplicity of infection (MOI) of 5000 vg / cell. B) iT progenitor cells were electroporated with a ribonucleoprotein (RNP) complex targeting the B2M target site in the placeholder TCR transgene cassette. C) iT progenitor cells were electroporated with an RNP complex targeting the B2M target site of the placeholder transgene and transduced with an rAAV encoding the A2M4 repair template at an MOI of 5000 vg / cell. D) Cells were electroporated with an RNP complex targeting the B2M target site of the placeholder transgene, transduced with an rAAV encoding the ADB01032_026A2M4 repair template at an MOI of 5000 vg / cell, and treated with 0.3 μM M3814. TCR expression was analyzed by flow cytometry 72 h after electroporation.

[0074] FIG15 shows the early 15F2_AAVS1 after CRISPR-Cas9-based gene editing. - / A2M10LP Quantification of placeholder TCR gene editing results in progenitor iT cells. A) Loss of A2M10Vβ13.2 placeholder TCR expression as a measure of CRISPR Cas9 knockout efficiency. B) Measurement of replacement of A2M10 Vβ13.2 placeholder TCR with A2M4 Vα24 TCR. C) Frequency of A2M4 Vα24 TCR expression in live progenitor iT cells. D) Frequency of A2M10Vβ13.2 placeholder TCR expression in live progenitor iT cells.

[0075] FIG. 16 shows early 16D5_AAVS1 after CRISPR-Cas9-based gene editing. A2M10LP / A2M10LP Expression of A2M10 placeholder TCR and A2M4TCR in progenitor iT cells. A) Cells were mock electroporated and transduced with A2M4rAAV-ADB01032_026 repair template at an MOI of 5000vg / cell. B) iT progenitor cells were electroporated with RNP complexes targeting the B2M target site in the placeholder TCR transgene cassette. C) iT progenitor cells were electroporated with RNP complexes targeting the B2M target site of the placeholder transgene and transduced with rAAV (ADB01032_026) encoding the A2M4 repair template at an MOI of 5000vg / cell. D) Cells were electroporated with RNP complexes targeting the B2M target site of the placeholder transgene, transduced with rAAV encoding the A2M4 repair template at an MOI of 5000vg / cell, and treated with 1μM M3814. TCR expression was analyzed by flow cytometry 72 h after electroporation.

[0076] FIG. 17 shows early 16D5_AAVS1 after CRISPR-Cas9-based gene editing. A2M10LP / A2M10LP Quantification of placeholder TCR gene editing results in progenitor iT cells. A) Loss of A2M10 placeholder TCR expression as a measure of CRISPRCas9 knockout efficiency. B) Measurement of replacement of A2M10 Vβ13.2 placeholder TCR with A2M4 Vα24 TCR. C) Frequency of A2M4 Vα24 TCR expression in live progenitor iT cells. D) Frequency of A2M10 Vβ13.2 placeholder TCR expression in live progenitor iT cells.

[0077] FIG. 18 shows the CD3 / CD28 activated late 15F2_AAVS1 after CRISPR-Cas9 based gene editing - / A2M10LPExpression of A2M10 placeholder TCR and A2M4 TCR in progenitor iT cells. A) Cells were mock electroporated and transduced with A2M4 rAAV-ADB01032_026 repair template at an MOI of 5000 vg / cell. B) iT progenitor cells were electroporated with RNP complexes targeting the B2M target site in the placeholder TCR transgene cassette. C) iT progenitor cells were electroporated with RNP complexes targeting the B2M target site of the placeholder transgene and transduced with rAAV encoding the A2M4-ADB01032_026 repair template at an MOI of 5000 vg / cell. D) Cells were electroporated with RNP complexes targeting the B2M target site of the placeholder transgene, transduced with rAAV encoding the A2M4 repair template at an MOI of 5000 vg / cell, and treated with 0.6 μM M3814. TCR expression was analyzed by flow cytometry 72 h after electroporation.

[0078] FIG. 19 shows the late stage of 15F2_AAVS1 in CD3 / CD28 activation after CRISPR-Cas9-based gene editing. - / A2M10LP Quantification of placeholder TCR gene editing results in progenitor iT cells. A) Loss of A2M10 placeholder TCR expression as a measure of CRISPR Cas9 knockout efficiency. B) Measurement of replacement of A2M10 Vβ13.2 placeholder TCR with A2M4 Vα24 TCR. C) Frequency of A2M4 Vα24 TCR expression in live progenitor iT cells. D) Frequency of A2M10 Vβ13.2 placeholder TCR expression in live progenitor iT cells. DETAILED DESCRIPTION

[0079] The present invention relates to the generation of immune cells expressing therapeutic antigen receptors such as T cell receptors (TCR). Immune cells are produced by iPSCs comprising heterologous expression cassettes, and the heterologous expression cassettes express "placeholders" to produce TCR. Producing the expression of TCR in cells avoids differentiation stagnation and allows the production of mature immune cells, such as CD3+T cells. Then, the heterologous expression cassette can be used as a "landing pad" for an expression construct containing a nucleotide sequence encoding a therapeutic antigen receptor to sensitize immune cells. The expression construct is inserted into the genome of the immune cell at the site of the heterologous expression cassette. For example, the expression construct can replace the heterologous expression cassette in the immune cell. The expression construct replaces the heterologous expression cassette in the immune cell, and the immune cell then expresses the therapeutic antigen receptor. The immune cells produced as described herein can be used for immunotherapy.

[0080] For example, methods described herein can be used to rapidly generate immune cells for treating cancer in patients. The therapeutic antigen receptors expressed by immune cells can be selected to be reactive with the cancer cells of the patient. Antigen receptors can be, for example, TCRs or other antigen receptors expressed by tumor infiltrating lymphocytes (TIL) obtained from the patient, or can be known to be reactive with tumor antigens identified as being expressed by cancer cells in the patient. An expression construct comprising a nucleotide sequence encoding an antigen receptor can be used to replace a heterologous expression cassette to produce immune cells that are specifically reactive with the cancer cells of the patient, and can be used to treat the cancer of the patient.

[0081] Immune cells suitable for use as described herein include T cells, such as αβ+ T cells, γδ+ T cells, mucosal-associated invariant type (MAIT) T cells, and NK T cells.

[0082] T cells (also called T lymphocytes) are white blood cells that play a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes by the presence of a T cell receptor (TCR) on the cell surface. There are several types of T cells, each with different functions.

[0083] T helper cells (T H cells) are called CD4 + T cells because they express the CD4 surface glycoprotein. + T cells play an important role in the adaptive immune system and help the activity of other immune cells by releasing T cell cytokines and helping to suppress or regulate immune responses. They are essential for the activation and growth of CD8+ T cells. C CD8+ T cells express CD8 surface glycoprotein. + T cells are used to destroy virus-infected cells and tumor cells. Most CD8 + T cells express TCRs that can recognize specific antigens displayed on the surface of infected or damaged cells by MHC class I molecules. Specific binding of TCRs and CD8 glycoproteins to antigens and MHC molecules results in T cell-mediated destruction of infected or damaged cells.

[0084] As described herein the single positive generated T cells may be double positive CD4+CD8+ T cells or CD4+ or CD8+ T cells. Preferably the T cells comprise CD8+ T cells.

[0085] Preferably, the T cells may include TCRαβ+T cells. The TCRαβ+T cells generated as described herein may be mature CD3+T cells. For example, the T cells may have an αβTCR+CD3+CD45+CD28+ phenotype.

[0086] In the methods described herein, immune cells are primed for therapeutic use by inserting an expression construct encoding a therapeutic TCR at the site of a heterologous expression cassette encoding a TCR-producing cell. For example, an expression construct encoding a therapeutic TCR can be substituted for a heterologous expression cassette encoding a TCR-producing cell.

[0087] TCR is a disulfide-linked membrane-anchored heterodimeric protein that contains highly variable α (alpha) and β (beta) chains or δ (delta) and γ (gamma) chains expressed as a complex with a constant CD3 chain molecule. T cells expressing these types of TCRs can be referred to as αβ (or α:β) T cells and δγ (or δ:γ) T cells.

[0088] TCR specifically binds to the major histocompatibility complex (MHC) of peptide fragments of the display target antigen on the cell surface. For example, TCR can specifically bind to the major histocompatibility complex (MHC) of peptide fragments of display tumor antigens on the surface of cancer cells. Alternatively, TCR can recognize specific antigens or peptides thereof that are independent of MHC presentation. T cells comprising such TCR can be produced according to the method of the present invention. MHC is a group of cell surface proteins that allow the acquired immune system to recognize 'exogenous' molecules. Proteins are degraded in cells and presented on the cell surface by MHC. MHC displaying 'exogenous' peptides (such as viruses or cancer-related peptides) is recognized by T cells with appropriate TCR, thereby promoting cell destruction pathways. MHC on the surface of cancer cells can display peptide fragments of tumor antigens (i.e., antigens present on cancer cells but not on corresponding non-cancerous cells). T cells that recognize these peptide fragments can exert CD8+ effects on cancer cells.

[0089] The generation and therapeutic TCR described herein are not naturally expressed by iPSC or immune cells described herein (i.e., the TCR is exogenous or heterologous). Suitable heterologous TCRs can specifically bind to class I or class II MHC molecules that display peptide fragments of target antigens. Generation and therapeutic TCRs can be synthetic or artificial TCRs (i.e., TCRs do not exist in nature).

[0090] Produce TCR and therapeutic TCR can be encoded by heterologous nucleic acid.The term "heterologous" refers to a polypeptide or nucleic acid that is exogenous to a specific biological system (such as a host cell) and is not naturally present in the system.Heterologous polypeptides or nucleic acids can be introduced into a biological system by artificial means (such as using recombinant technology).For example, a heterologous nucleic acid encoding a polypeptide can be inserted into a suitable expression construct, which is then used to transform a host cell to produce a polypeptide.Heterologous polypeptides or nucleic acids can be synthetic or artificial, or can be present in different biological systems (such as different species or cell types).Endogenous polypeptides or nucleic acids are natural to a specific biological system (such as a host cell) and are naturally present in the system.Recombinant polypeptides are expressed by heterologous nucleic acids, and the heterologous nucleic acids have been introduced into cells by artificial means (such as using recombinant technology).Recombinant polypeptides can be the same as polypeptides naturally present in cells, or can be different from polypeptides naturally present in the cells.

[0091] The coding sequence for a TCR (e.g., generating a TCR or therapeutic antigen receptor) may comprise coding sequences for the alpha and beta chains or delta and gamma chains separated by a nucleotide sequence encoding a self-cleaving peptide (e.g., a 2A peptide). This allows expression of both chains according to stoichiometry in a single transcript.

[0092] Heterologous expression cassette is a recombinant nucleic acid incorporated into the genome of immune cells and their precursors. Heterologous expression cassettes support the generation of mature immune cells by allowing the expression of TCR to be produced. For example, the expression of TCR allows progenitor cells to differentiate into T cells. After mature immune cells are produced, the heterologous expression cassette forms a "landing pad", which allows the expression construct to replace the heterologous expression cassette at the same site in the genome. As described below, the expression cassette can include any suitable nucleic acid sequence. Preferred heterologous expression cassettes can be excised with a single guide RNA to completely remove the production of TCR.

[0093] TCR is expressed by immune cells and their precursors during TCR production. In cells lacking TCR expression, differentiation into immune cells is stagnant. The expression of producing TCR can promote the production of mature immune cells such as T cells. For example, the expression of producing TCR in immune cells can induce or promote the surface expression of CD3, and allow differentiation into lymphocytes such as CD3+T cells to produce lineages. After the differentiated CD3+ immune cells have been produced, the therapeutic antigen receptor can be inserted into the site where the TCR is produced. For example, the production of TCR can be replaced by a therapeutic antigen receptor in the cell.

[0094] Suitable generating TCRs include any TCR that supports T cell differentiation and surface expression of CD3 and prevents differentiation arrest. Unlike therapeutic antigen receptors, generating TCRs are not patient specific and do not mediate any therapeutic effect of the patient's immune cells.

[0095] In some embodiments, the generation of TCRs may lack binding activity. For example, the generation of TCRs may be functionally inert and may lack TCR function except for promoting T cell differentiation and surface CD3 expression. This can be used, for example, to reduce the need for separating or purifying T cells expressing therapeutic antigen receptors after the replacement generation of TCRs. Suitable functionally inert generation of TCRs may, for example, lack one or two TCR variable regions. For example, the generation of TCRs may lack alpha chain variable regions and / or beta chain variable regions.

[0096] In some embodiments, the generated TCR may bind to a class 1 MHC that displays an antigen fragment that has no clinical relevance. For example, the generated TCR may show no binding or substantially no binding to a tumor antigen or other clinically relevant antigen, and may not bind to a patient's cancer cells. In some embodiments, the generated TCR may be engineered to reduce or eliminate its affinity or avidity for an antigen.

[0097] Suitable generated TCRs may comprise various combinations of alpha and beta chains or variants thereof, or gamma and delta chains or variants thereof.Generated TCRs may be human or non-human, such as murine TCRs. For example, a TCR can be generated that comprises or consists of: (i) full-length α and β chains; (ii) α and β constant domains (TRAC (P01848-1) and TRBC (P01850-1)); (iii) a single-chain αβ TCR (e.g., a TCR having an α chain and a β chain connected by a peptide linker); (iv) a β chain and a chimeric chain comprising the variable domain and constant domain of the α chain fused to the transmembrane domain and cytoplasmic domain of the pre-α chain; (v) a full-length β chain and a full-length pre-α chain; (vi) a full-length β chain and a truncated pre-α chain with 48 aa deleted at the C-terminus (Δ48); (vii) a fragment of the β chain comprising or consisting of residues 125-176 (P01850-1; TRBC1_human aa 125-176; ) and a fragment of the pre-alpha chain comprising or consisting of residues 126 to 281 (PTCRA_human aa 126-281 (A0A087WTE9-1)); or (viii) the constant domain of the beta chain and the full-length pre-alpha chain.

[0098] In some preferred embodiments, the generated TCR may comprise or consist of: (i) a full-length α chain and a β chain or (ii) a full-length β chain and a full-length pre-α chain.

[0099] In other preferred embodiments, the TCR produced may comprise or consist of: (i) a β chain and a chimeric chain comprising the variable domain, constant domain and transmembrane domain of an α chain fused to the cytoplasmic domain of a pre-α chain, or (ii) a β chain and a chimeric chain comprising the variable domain and constant domain of an α chain fused to the transmembrane domain and cytoplasmic domain of a pre-α chain.

[0100] The amino acid and encoding nucleotide sequences of suitable alpha chains, pre-alpha chains and beta chains and domains thereof are well known in the art.

[0101] TCRs suitable for use as described herein are readily available in the art and include MAGE-A10 αβ TCR clone 796 (SEQ ID NOs: 14 to 17; SEQ ID NO: 60); MR1 TCR MC.7.G5 clone-αβ TCR (TRAV38.2 / DV8 TRAJ31 α-chain; TRBV25.1TRBJ2.3 β-chain) (Crowther et al., 2020 Nature Immunology 21 178-185; constant NKT αβ TCR (Vα24-Jα18 paired with Vβ11); γδ TCR Vγ5Vδ1 or Vγ1Vδ4 (Ribot et al., (2021) Nature Rev Immunology 21 221-232); γδ TCR Vγ9JPVδ2 (Ravens et al., (2018) Frontiers in Immunology 9 510; DiLorenzo et al., (2019) Sci Data 6 115; Xu et al., 2021 Cell Mol Immunol. 2021 Feb; 18(2):427-439); and HLA-E restricted TCRs against viral antigens such as CMV and HIV (Yang et al., (2021) Sci Immunol. 6 57; Pietra et al., (2003) PNAS USA 100(19):10896-10901). In some preferred embodiments, the α chain amino acid sequence of SEQ ID NO: 14 and the α chain amino acid sequence of SEQ ID NO: 14 can be used. The alpha chain may be encoded by the nucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 66, and the beta chain may be encoded by the nucleotide sequence of SEQ ID NO: 17 or SEQ ID NO: 71.

[0102] Suitable nucleotide sequences are well known in the art. The heterologous expression cassette may include one or more nucleic acids encoding TCR. The heterologous nucleic acid encoding TCR may encode all subunits of the receptor. Preferably, the chain generating TCR is expressed in a single transcript. For example, the nucleic acid encoding TCR may include a first nucleotide sequence encoding TCR alpha chains and a second nucleotide sequence encoding TCR beta chains, or a first nucleotide sequence encoding TCR delta chains and a second nucleotide sequence encoding TCR gamma chains. The encoding nucleic acid may further include a nucleotide sequence encoding a 3' poly (A) sequence. Suitable nucleotide sequences encoding poly (A) sequences are shown in SEQ ID NO:4, SEQ ID NO:10, and SEQ ID NO:40.

[0103] In some embodiments, the heterologous expression cassette may comprise one or more nucleic acids encoding a CD3 chimeric fusion receptor rather than generating a TCR.

[0104] The self-cleavage peptide coding sequence can be located between the first nucleotide sequence encoding TCR α or TCR δ chain and the second nucleotide sequence encoding TCR β chain or TCR γ chain. The self-cleavage peptide causes the cleavage of the nascent peptide chain by ribosome jumping during translation, and separates the TCR chain. Suitable 2A peptides may include T2A peptides, P2A peptides, E2A peptides, and F2A peptides (Poddar et al., (2018) the same; Kim et al., (2011) "Public Library of Science Comprehensive (PLoS ONE)" 6, e18556). Preferred 2A peptides may include SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:69 amino acid sequences. The self-cleavage peptide coding sequence may include SEQ ID NO:2, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:70 and SEQID NO:80 nucleotide sequences.

[0105] The nucleic acid encoding the furin cleavage site can be located adjacent to the self-cleaving peptide coding sequence. This may help to remove the self-cleaving peptide residues from the TCR chain. Suitable furin cleavage sites and coding sequences are shown in SEQ ID No11, 12, 24 to 27 and 68.

[0106] The expression cassette may further comprise a promoter operably connected to the coding sequence for producing TCR. The promoter can drive the expression of the production TCR in immune cells. Suitable promoters include constitutive promoters, such as SV40, CMV, UBC, EF1A, EF1AS, PGK, JeT, MND or CAGG promoters or variants thereof. The nucleotide sequence of a suitable EF1A promoter is shown in SEQ ID NO:3, 36 and 65. Examples of nucleotide sequences of the expression cassettes for producing TCR by A2M10 are shown in SEQ ID NO:58 and 76.

[0107] The heterologous expression cassette comprises a targeting site. The targeting site is a nucleotide sequence that mediates the insertion of an expression construct at the site of the expression cassette. For example, the targeting site can mediate the replacement of a heterologous expression cassette in the immune cell genome with an expression construct. In certain embodiments, the targeting site can be positioned upstream of a constitutive promoter in the heterologous expression cassette. Preferably, the targeting site can be positioned at the 5' end of the cassette. In other embodiments, the targeting site can be positioned within a coding sequence for generating a TCR.

[0108] In some embodiments, the heterologous expression cassette may comprise 5' and 3' targeting sites. For example, the heterologous expression cassette may be cut at the 5' and 3' targeting sites and excised from the genome of the immune cell. In some embodiments, the targeting site or the nucleotide sequence of the 5' and 3' targeting sites is unique in the genome of the immune cell.

[0109] In some embodiments, the 5' targeting site may be positioned upstream of a constitutive promoter in a heterologous expression cassette. Preferably, the 5' targeting site may be positioned at the 5' end of the cassette.

[0110] In some embodiments, the 3' targeting site may be positioned downstream of the coding sequence or at the 3' end of the coding sequence. Preferably, the 3' targeting site may be positioned at the 3' end of the cassette.

[0111] One of the 5' and 3' targeting sites may be located within the coding sequence used to generate the TCR.

[0112] The selection of 5' and 3' targeting sites can depend on the technology selected for the replacement expression cassette. For example, suitable 5' and 3' targeting sites can include CRISPR guide RNA recognition sequences for CRISPR-mediated replacement, loxP sites for CRE-LOXP-mediated replacement, FRT sites for FLP-FRT-mediated replacement; and recognition sites for site-specific nucleases such as transcription activator-like effector nucleases (TALENs).

[0113] Any suitable technique can be used to insert the expression construct into the cell genome at the site of the expression cassette. In some preferred embodiments, the expression cassette can be replaced with an expression construct using CRISPR-mediated alternative techniques. For example, 5' and 3' targeting sites can include CRISPR guide RNA recognition sequences. Suitable guide RNA recognition sequences can, for example, contain 19 to 21 nucleotides. Preferably, the guide RNA recognition sequence is unique in the immune cell genome to avoid off-target effects. Examples of suitable guide RNA recognition sequences include SEQ ID NO: 5 to 9. Methods for designing guide RNA recognition sequences suitable for CRISPR-mediated alternatives are well-known in the art.

[0114] In some embodiments, the targeting site or one of the 5' and 3' targeting sites, preferably the 3' targeting site, can be located within the coding sequence for generating the TCR, for example, within the TCR chain constant region coding sequence, such as the TCR alpha chain constant region coding sequence (TRAC) or the TCR beta chain constant region coding sequence (TRBC). An example of a preferred 5' targeting site comprising a sequence from the MAGE-A10 c796 TCR alpha chain (TRAC) coding sequence is shown in SEQ ID NO: 13. The corresponding 3' targeting site is located within the MAGE-A10 c796 TCR alpha chain coding sequence of the expression cassette (see SEQ ID NO: 16).

[0115] In other embodiments, the targeting site or one of the 5' and 3' targeting sites, preferably the 3' targeting site, may comprise a nucleotide sequence from the locus into which the expression cassette is inserted.

[0116] In some embodiments, the 5' and 3' targeting sites can comprise the same nucleotide sequence. This can facilitate, for example, the removal of a heterologous expression cassette using a single guide RNA. For example, the same targeting site can be located at both the 5' and 3' ends of the expression cassette. For example, the expression cassette can comprise a TRAC or TRBC sequence at its 5' and 3' ends.

[0117] An example of a nucleotide sequence for insertion into an A2M10 TCR-producing plasmid is shown in SEQ ID NO:59.

[0118] The heterologous expression cassette can be incorporated into the genome of the immune cell. In certain embodiments, the heterologous expression cassette can be incorporated into the genome of the immune cell in the locus comprising an endogenous promoter. For example, the heterologous expression cassette can be integrated into the exon of the gene in the locus or next to the exon, preferably integrated into the last exon of the gene in the locus. The integration retains the natural reading frame of the gene so that after replacing the heterologous expression cassette with an expression construct as described herein, the expression of the therapeutic antigen receptor is driven by the endogenous promoter. Suitable loci can be active in differentiated T cells, and can include TRAC, PTPRC, EEF1A1, CD3E, CD3D, CD3G, CD8A and CD2.

[0119] In other embodiments, a heterologous expression cassette can be incorporated into the genome of an immune cell within a safe harbor locus. This allows expression of a therapeutic antigen receptor to be driven by a constitutive promoter contained in the expression construct. Suitable expression constructs can, for example, comprise a nucleic acid encoding a poly(A) sequence and a constitutive promoter. Suitable safe harbor loci include AAVS1 and are shown in Table 1.

[0120] After inserting the expression construct at the site of the expression cassette, the therapeutic antigen receptor is expressed by the immune cell. For example, the receptor can be expressed after replacing the expression cassette with the expression construct.

[0121] The therapeutic antigen receptor mediates the therapeutic effect of immune cells. Preferably, the therapeutic antigen receptor binds to the patient's cancer cells. For example, the therapeutic T antigen receptor can specifically bind to class I or class II MHC molecules, which display peptide fragments of tumor antigens expressed by cancer cells of cancer patients. In some embodiments, the therapeutic T antigen receptor can recognize a target antigen or a peptide fragment of a target antigen on a cancer cell that is independent of MHC presentation. Tumor antigens expressed by cancer cells of cancer patients can be identified using standard techniques. Preferred tumor antigens include NY-ESO1, PRAME, α-fetoprotein (AFP), MAGE A4, MAGE A1, MAGE A10 and MAGE B2, most preferably NY-ESO-1, MAGE-A4 and MAGE-A10.

[0122] In some embodiments, a patient's tumor antigen can be identified and a therapeutic antigen receptor that binds to the tumor antigen selected for expression in the construct.

[0123] In some embodiments, the therapeutic antigen receptor can be a chimeric antigen receptor (CAR). CAR is an artificial receptor engineered to contain an immunoglobulin antigen binding domain, such as a single-chain variable fragment (scFv). CAR can, for example, comprise an scFv fused to the TCR CD3 transmembrane region and the endodomain. ScFv is a heavy chain (VH ) and light chain (V L ) can be connected to a short linker peptide of about 10 to 25 amino acids (Huston JS et al., Proc Natl Acad Sci USA 1988; 85(16):5879–5883). The linker can be rich in glycine to improve flexibility and rich in serine or threonine to improve solubility, and V H The N-terminal and V L The C-terminus of the scFv may be connected, or vice versa. There may be a signal peptide before the scFv to guide the protein to the endoplasmic reticulum and then to the T cell surface. In CAR, the scFv can be fused to the TCR transmembrane and internal domains. A flexible spacer may be included between the scFv and the TCR transmembrane domain to allow variable orientation and antigen binding. The internal domain is the functional signal transmission domain of the receptor. The internal domain of the CAR may include, for example, an intracellular signaling domain from a CD3 ζ chain or from a receptor such as CD28, 41BB or ICOS. CAR may include multiple signaling domains, such as, but not limited to, CD3z-CD28-41BB or CD3z-CD28-OX40.

[0124] CAR can specifically bind to tumor-specific antigens expressed by cancer cells. For example, T cells can be modified to express CARs that specifically bind to tumor antigens expressed by cancer cells of a specific cancer patient. Tumor antigens expressed by cancer cells of a cancer patient can be identified using standard techniques.

[0125] In other embodiments, the therapeutic antigen receptor may be a NK cell receptor (NKCR).

[0126] In other embodiments, the therapeutic antigen receptor can be a T cell receptor (TCR). TCR is described elsewhere herein and can include αβTCR heterodimers and γδTCR heterodimers. Suitable heterologous TCRs can specifically bind to class I or class II MHC molecules that display peptide fragments of target antigens. For example, T cells can be modified to express heterologous TCRs that specifically bind to class I or class II MHC molecules that display peptide fragments of tumor antigens expressed by cancer cells of cancer patients. Tumor antigens expressed by cancer cells of cancer patients can be identified using standard techniques. Preferred tumor antigens include NY-ESO1, PRAME, α-fetoprotein (AFP), MAGE A4, MAGE A1, MAGE A10, and MAGE B2, most preferably NY-ESO-1, MAGE-A4, and MAGE-A10.

[0127] In some preferred embodiments, the heterologous TCR can specifically bind to HLA-A*02:01 displaying the MAGEA4 peptide fragment GVYDGREHTV.

[0128] Suitable therapeutic TCRs may include unconventional TCRs, such as MHC-independent TCRs that recognize non-peptide antigens displayed by monomorphic antigen presenting molecules, such as CD1 and MR1; NKT cell TCRs and intraepithelial lymphocyte (IEL) TCRs. In some embodiments, therapeutic TCRs may recognize target antigens or peptide fragments of target antigens that are independent of MHC presentation on cancer cells.

[0129] Suitable therapeutic TCR includes TCR from patient.For example, therapeutic TCR can be TCR from immune cells such as tumor infiltrating lymphocytes (TIL) obtained from donor individuals.For example, the patient's tumor can be analyzed to identify the tumor antigen expressed by the cancer cells in the tumor. TCR with responsiveness to the identified tumor antigen can be identified, and inserted into the expression construct, to be used as therapeutic TCR as described herein. In other embodiments, the TCR expressed by immune cells such as tumor infiltrating lymphocytes (TIL) obtained from the patient can be sequenced and cloned into the expression construct. For example, as described herein, various TCR libraries from the patient can be cloned into the expression construct, for insertion into immune cells. The art has established the TCR encoding sequence for obtaining immune cells such as tumor infiltrating lymphocytes (TIL) obtained from the patient and inserting it into the appropriate technology in the expression construct. The target tumor antigen from the TCR of TIL can be identified, or it can also remain unidentified. The donor individual may be the same person as the recipient individual to whom the immune cells will be administered following generation as described herein, ie, the therapeutic TCRs may be derived from the patient to whom the immune cells are administered.

[0130] In some embodiments, therapeutic TCRs can be engineered to increase their affinity or avidity for tumor antigens (i.e., affinity-enhanced TCRs). Relative to naturally occurring TCRs, affinity-enhanced TCRs can include one or more mutations, for example, one or more mutations in the hypervariable complementary determining regions (CDRs) of the variable regions of the TCR α and β chains. These mutations can increase the affinity of the TCR for MHCs displaying peptide fragments of tumor antigens expressed by cancer cells. Suitable methods for generating affinity-enhanced TCRs include screening libraries of TCR mutants using phage or yeast display, and such suitable methods are well known in the art (see, e.g., Robbins et al., J Immunol (2008) 180(9):6116; San Miguel et al., (2015) Cancer Cell 28(3)281-283; Schmitt et al., (2013) Blood 122 348-256; Jiang et al., (2015) Cancer Discovery 5 901).

[0131] An example of an amino acid sequence of A2M4 TCR alpha is shown in SEQ ID NO: 79, and an example of an amino acid sequence of A2M4 TCR beta is shown in SEQ ID NO: 82. An example of an amino acid sequence of a therapeutic TCR is shown in SEQ ID NO:62.

[0132] The expression construct is a recombinant nucleic acid incorporated into the genome of an immune cell at the site of a heterologous expression cassette. For example, an expression construct can replace a heterologous expression cassette. The expression construct comprises a coding sequence for a therapeutic TCR. The coding sequence for a therapeutic TCR may, for example, comprise a first nucleotide sequence encoding a TCR alpha chain and a second nucleotide sequence encoding a TCR beta chain or a first nucleotide sequence encoding a TCR gamma chain and a second nucleotide sequence encoding a TCR delta chain. Examples of suitable first nucleotide sequences encoding ADB959 TCR alpha are shown in SEQ ID NO:33 and SEQ ID NO:38. Examples of suitable second nucleotide sequences encoding ADB959 TCR beta are shown in SEQ ID NO:32 and SEQ ID NO:37. Examples of suitable first nucleotide sequences encoding A2M4 TCR alpha are shown in SEQ ID NO:78, and examples of suitable second nucleotide sequences encoding A2M4 TCR beta are shown in SEQ ID NO:81.

[0133] The first and second nucleotide sequences may be located in a single open reading frame and may be separated by a third nucleotide sequence encoding a self-cleaving peptide, such as a 2A peptide and / or a furin linker. Suitable self-cleaving peptides are described in more detail above.

[0134] An example of an amino acid sequence encoded by the expression construct (PTPRC exon 33_T2A_ADB959_TCRβ_P2A_TCRα) is shown in SEQ ID NO: 34. The amino acid sequence includes T2A and P2A sequences to separate the ADB959 TCR chain and the PTPRC exon sequence.

[0135] In some embodiments, the expression construct may further comprise a promoter operably linked to a coding sequence for a therapeutic antigen receptor. The promoter may drive expression of the therapeutic antigen receptor in immune cells. Suitable promoters include constitutive promoters such as SV40, CMV, UBC, EF1A, EF1AS, PGK, JeT, MND or CAGG promoters or variants thereof. The nucleotide sequence of a suitable EF1A promoter is shown in SEQ ID NO:3 and SEQ ID NO:36.

[0136] The expression construct can be inserted into the immune cell genome at the same site as the heterologous expression cassette. For example, the heterologous expression cassette can be replaced with the expression construct in the immune cell. This sensitizes the immune cell for therapeutic use of the individual. Preferably, the heterologous expression cassette is completely excised so that no sequence from the heterologous expression cassette remains in the immune cell after replacement. Any suitable technique can be used to achieve the replacement of the heterologous expression cassette with an expression construct.

[0137] An example of a nucleotide sequence of a plasmid for inserting the A2M4 therapeutic TCR is shown in SEQ ID NO:61.

[0138] Suitable techniques include HR-mediated gene replacement techniques such as CRISPR / Cas9-based techniques and recombinase-mediated gene replacement techniques such as Cre-Lox, FLP-FRT or phiC31 integrase techniques. Suitable techniques are well known in the art (see, for example, Yamamoto et al., Chromosoma. (2018) 127(4): 405-420; Sakuma et al., (2016) Nat Protoc 11(1) 118-133).

[0139] In some preferred embodiments, the expression cassette can be replaced by HR-mediated target gene replacement. For example, the heterologous expression cassette can be replaced by a method comprising:

[0140] introducing into the immune cell a nucleic acid molecule, such as a DNA molecule, comprising an expression construct flanked by 5' and 3' homology arms, wherein the 5' and 3' homology arms are complementary to nucleotide sequences at the 5' and 3' ends of the heterologous expression cassette and / or genomic sequences flanking the heterologous expression cassette,

[0141] The expression construct is allowed to replace the expression cassette in the genome of the immune cell.

[0142] After cleavage of the heterologous expression cassette at the 5' and 3' targeting sites, the homology arms mediate replacement of the heterologous expression cassette with the expression construct. Suitable homology arms may comprise a sequence of 300 to 500 nucleotides complementary to the nucleotide sequence of the heterologous expression cassette and / or a genomic sequence flanking the heterologous expression cassette, the genomic sequence being 5' or 3' to the 5' and 3' targeting sites, respectively, such that the homology arms are complementary to the sequence at the locus or safe harbor locus after removal or excision of the heterologous expression cassette sequence between the targeting sites.

[0143] In some embodiments, HR-mediated target gene replacement can be mediated by programmable nucleases, such as site-specific nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases, or RNA-guided nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR) nucleases.

[0144] Zinc finger nucleases (ZFNs) comprise one or more Cys2-His2 zinc finger DNA binding domains and a cleavage domain (i.e., nuclease). Conventional techniques can be used to engineer the DNA binding domain to recognize and bind to any nucleic acid sequence (see, e.g., Qu et al., (2013) Nucleic Acids Research (Nucl Ac Res) 41(16):7771-7782). The use of ZFNs to introduce mutations into a target gene is well known in the art (see, e.g., Beerli et al., Nat. Biotechnol., 2002; 20: 135-141; Maeder et al., Mol. Cell. 2008; 31: 294-301; Gupta et al., Nat. Methods 2012; 9: 588-590) and engineered ZFNs are commercially available (Sigma-Aldrich, St. Louis, MO).

[0145] Transcription activator-like effector nuclease (TALEN) comprises a non-specific DNA-cutting nuclease fused to a DNA binding domain, and the DNA binding domain comprises a series of modular TALEN repeats linked together to recognize continuous nucleotide sequences. The use of TALEN targeted nucleases is well known in the art (e.g., Joung and Sander (2013) Nat Rev Mol Cell Biol. 14:49-55; Kim et al., Nat Biotechnol. (2013); 31:251–258. Miller JC et al., Nat Biotechnol. (2011) 29:143-148. Reyon D et al., Nat Biotechnol. (2012); 30:460–465).

[0146] Meganucleases are endodeoxyribonucleases characterized by a large recognition site (a double-stranded DNA sequence of 12 to 40 base pairs); thus this site usually occurs only once in any given genome (see, e.g., Silva et al., (2011) Curr Gene Ther 11(1): 11-27).

[0147] CRISPR targeting nucleases (e.g., Cas9) are mixed with guide RNAs (gRNAs) to cleave genomic DNA in a sequence-specific manner. The crRNA and tracrRNA of the guide RNA can be used alone or can be combined into a single RNA to enable site-specific mammalian genome cleavage at the 5' and 3' targeting sites of the expression cassette. For example, as a way to introduce transgenes, the use of the CRISPR / Cas9 system to introduce double-strand breaks into a locus is well known in the art (see, e.g., Cader et al., Nature Immunology 2016 17(9) 1046-1056, Hwang et al., (2013) Nature Biotechnology 31:227-229; Xiao et al., (2013) Nucleic Acids Res 1-11; Horvath et al., Science (2010) 327:167-170; Jinek M et al., Science (2012) 337:816-821; Cong L et al., Science (2013) 339:819-823; Jinek M et al., (2013) eLife 2:e00471; Mali P et al., (2013) Science, 339:823-826; Qi et al., (2014) Science 339:834-835; LS et al. (2013) Cell, 152:1173-1183; Gilbert LA et al. (2013) Cell, 154:442-451; Yang H et al. (2013) Cell, 154:1370-1379; and Wang H et al. (2013) Cell, 153:910-918).

[0148] In some preferred embodiments, the targetable nuclease is a Cas endonuclease, which is expressed in immune cells in combination with a guide RNA that targets the Cas endonuclease to cleave the heterologous expression cassette at the 5' and 3' targeting sites.

[0149] Preferably, HR-mediated target gene replacement is mediated by CRISPR / Cas9. For example, a DNA double-strand break (DSB) at the target site can be induced by the CRISPR / Cas9 system, and the repair of DSB can introduce the expression construct into the cell genome at the target site, or rAAV vectors can be used to introduce nucleic acids (AAV-mediated gene editing; Hirsch et al., 2014 "Molecular Biology Methods (Methods Mol Biol)" 1114 291-307). For example, a heterologous expression cassette can be replaced by a method comprising the following:

[0150] introducing into the immune cell a nucleic acid molecule, such as a DNA molecule, comprising an expression construct flanked by 5' and 3' homology arms, wherein the 5' and 3' homology arms are complementary to nucleotide sequences at the 5' and 3' ends of the heterologous expression cassette and / or genomic sequences flanking the heterologous expression cassette, and

[0151] Introducing CRISPR / Cas9 targeting 5' and 3' targeting sites into immune cells,

[0152] The expression construct is allowed to replace the expression cassette in the genome of the immune cell.

[0153] Suitable homology arms are as described above, and may comprise a sequence of 300-500 nucleotides that is complementary to the nucleotide sequences at the 5' and 3' ends of the heterologous expression cassette and / or the genomic sequences flanking the heterologous expression cassette.

[0154] Suitable homology arms for DNA molecules in exon 33 of PTPRC or targeting vectors for expression cassettes are shown in SEQ ID NOs: 22 and 23. Suitable homology arms for DNA molecules in intron 1 of PPP1R12C (AAVS1) or targeting vectors for expression cassettes are shown in SEQ ID NOs: 35 and 41. Other suitable homology arms are shown in SEQ ID NOs: 64 and 74 or SEQ ID NOs: 77 and 83 or as described elsewhere herein.

[0155] The DNA molecule comprising the expression construct can be a single-stranded DNA molecule. Suitable single-stranded DNA molecules can be contained in a recombinant adeno-associated virus (rAAV) vector. By transfecting cells with rAAV vectors, single-stranded DNA molecules can be introduced into immune cells.

[0156] Suitable targeting sites such as 5' and 3' targeting sequences can include nucleotide sequences complementary to the guide RNA of CRISPR / Cas9. Suitable sequences can, for example, consist of 17 to 24 nucleotides. The targeting site can further include additional nucleotide sequences flanked by the complementary nucleotide sequence. When the heterologous expression cassette is removed, this may be required to improve efficacy. For example, at the 5' and 3' ends of the complementary nucleotide sequence, the targeting site can include additional 1 to 15 nucleotides, preferably about 12 nucleotides.

[0157] In some embodiments, the 3' targeting site can be a nucleotide sequence located within the coding sequence for the constant region of the TCR alpha chain within the expression cassette. For example, the 3' targeting site can be the 3' end of the sequence encoding the constant region of the TCR alpha chain. The 5' targeting site has the same nucleotide sequence as the 3' targeting site, i.e., the 5' targeting site can be a copy of the 3' end of the sequence encoding the constant region of the TCR alpha chain. The 5' targeting site can be located upstream of the promoter within the expression cassette.

[0158] CRISPR / Cas9 can be introduced directly into cells as proteins and gRNA, for example, in lipid nanoparticles, or can be introduced as nucleic acids encoding CRISPR / Cas9 (such as mRNA, plasmids or viral vectors) and then expressed in cells. Nucleic acids encoding CRISPR / Cas9 can be introduced into immune cells by any convenient method such as RNP electroporation.

[0159] Because they have the same sequence, a single guide RNA can target CRISPR / Cas9 to both the 5' and 3' targeting sites, thereby cleaving the expression cassette at its 5' and 3' ends and excising it from the genome. After the sequence of the expression cassette between the 5' and 3' targeting sites is excised, the 5' and 3' homology arms (which are complementary sequences at the locus outside the targeting site or at the safe harbor locus) mediate the incorporation of the expression construct into the locus.

[0160] Suitable guide RNA sequences for CRISPR-Cas9-mediated gene replacement can be designed using standard techniques. For example, suitable guide RNA sequences targeting TRAC1 include SEQ ID NOs: 5 and 6. Suitable guide RNA sequences targeting the AAVS1 safe harbor sequence include SEQ ID NO: 7. Suitable guide RNA sequences targeting exon 2 of the B2M sequence include SEQ ID NO: 8. Suitable guide RNA sequences targeting the PTPRC sequence include SEQ ID NO: 9.

[0161] The methods described herein may further include, for example, reducing the expression of endogenous TCR in the cell or silencing it by inactivating endogenous TCR genes or endogenous RAG1 or RAG2 genes. For example, the method may further include inactivating endogenous TCR alpha (TRAC) chain genes or TCR beta (TRBC1 or 2) chain genes or endogenous RAG1 or RAG2 genes. This can be used to reduce or prevent the off-target toxicity of immune cells. Endogenous genes can be inactivated in immune cells such as IPSCs or progenitor cells. For example, before incorporating a heterologous expression cassette, endogenous genes can be inactivated in IPSCs.

[0162] Any suitable technique can be used to inactivate the endogenous TCR gene. Conveniently, the endogenous TCR gene is inactivated while the heterologous expression cassette is replaced. In some preferred embodiments, the CRISPR / Cas9 replacement expression cassette is used to target the sequence in the cassette encoding the constant region of the TCR α chain. CRISPR / Cas9 can also target the sequence in the endogenous gene encoding the constant region of the TCR α chain. This may introduce one or more inactivating mutations into the endogenous TCR α chain constant region (TRAC) gene.

[0163] The methods described herein may further include, for example, reducing the expression of class II transcription activator (CIITA) and / or beta-2-microglobulin (B2M) by inactivating endogenous B2M or CIITA genes or silencing them. Endogenous genes can be inactivated in immune cells such as IPSC or progenitor cells. This may help to reduce alloreactive effects and improve the persistence of immune cells in vivo. In certain embodiments, the methods described herein may further include expressing heterologous B2M-HLA-E (mBE) and B2M-HLA-G (mBG) fusion proteins in immune cells. A construct comprising a heterologous nucleic acid encoding a fusion protein operably connected to a suitable promoter may be inserted into immune cells such as IPSC or progenitor cells. This may help to protect cells from allogeneic NK cell-mediated lysis.

[0164] The immune cells may display expression of a therapeutic TCR or multiple therapeutic TCRs but not expression of endogenous TCRs.

[0165] Immune cells comprising a heterologous expression cassette can be generated by directed differentiation from induced pluripotent stem cells (iPSCs). For example, a method for generating an immune cell comprising a heterologous expression cassette can comprise:

[0166] (i) transfecting an iPSC with a nucleic acid comprising a heterologous expression cassette such that the cassette is integrated into the genome of the iPSC,

[0167] Wherein the expression cassette comprises:

[0168] (a) a coding sequence for producing a T cell receptor (TCR),

[0169] (b) a constitutive promoter operably linked to the coding sequence, and

[0170] (c) target site, and

[0171] (ii) differentiating the iPSCs into immune cells comprising the expression cassette.

[0172] In some embodiments, the targeting site can be a 5' targeting site, and the cassette can further comprise a 3' targeting site.

[0173] The heterologous expression cassette can be integrated into the target locus of iPSCs by a method comprising:

[0174] introducing into the iPSC a nucleic acid molecule, such as a DNA molecule, comprising an expression cassette flanked by 5' and 3' homology arms, wherein the 5' and 3' homology arms are complementary to nucleotide sequences flanking the integration site in the target locus, and

[0175] Introducing CRISPR / Cas9 targeted to the integration site in the target locus into immune cells,

[0176] The expression cassette is allowed to integrate into the genome of the immune cell at the integration site in the target locus.

[0177] Suitable homology arms are as described above, and may comprise a sequence of 300 to 500 nucleotides that is complementary to the nucleotide sequence flanking the integration site in the target locus.Homology arms may be designed for any target locus using standard techniques.

[0178] Suitable target loci are described above and shown in Table 1. In some embodiments, the heterologous expression cassette can be integrated into exon 33 of PTPRC. Suitable homology arms for DNA molecules or targeting vectors for exon 33 of PTPRC are shown in SEQ ID NOs: 18 and 19. In other embodiments, the heterologous expression cassette can be integrated into intron 1 of PPP1R12C (AAVS1). Suitable homology arms for DNA molecules or targeting vectors for intron 1 of PPP1R12C (AAVS1) are shown in SEQ ID NOs: 20 and 21.

[0179] The DNA molecule comprising the heterologous expression cassette can be a single-stranded DNA molecule. Suitable single-stranded DNA molecules can be contained in a recombinant adeno-associated virus (rAAV) vector. By transfecting cells with rAAV vectors, single-stranded DNA molecules can be introduced into immune cells.

[0180] Induced pluripotent stem cells (iPSCs) are pluripotent stem cells derived from non-pluripotent, fully differentiated donors or previous cells. iPSCs are capable of self-renewal in vitro and exhibit an undifferentiated phenotype, as well as potentially being able to differentiate into any fetal or adult cell type in any of the three germ layers (endoderm, mesoderm, and ectoderm). The colony of iPSCs can be cloned, i.e., genetically identical cells are descendants of a single common ancestral cell. iPSCs may express one or more of the following pluripotency-related markers: POU5f1 (Oct4), Sox2, alkaline phosphatase, SSEA-3, Nanog, SSEA-4, Tra-1-60, KLF4, and c-myc, preferably one or more of POU5f1, NANOG, and SOX2. iPSCs may lack markers associated with specific differentiation fates, such as Bra, Sox17, FoxA2, ​​αFP, Sox1, NCAM, GATA6, GATA4, Hand1, and CDX2. Specifically, iPSCs may lack markers associated with endoderm fate.

[0181] Preferably, the iPSCs are human iPSCs (hiPSCs).

[0182] In some embodiments, iPSCs can be gene-edited, for example to inactivate or delete HLA genes or other genes associated with immunogenicity or GVHD.

[0183] IPSC can be derived or reprogrammed from donor cells, which can be somatic cells or other previous cells obtained from sources such as donor individuals. Donor cells can be mammalian cells, preferably human cells. Suitable donor cells include adult fibroblasts and blood cells, such as peripheral blood cells, such as HPC or monocytes. As described herein, suitable donor cells for reprogramming into iPSC can be obtained from donor individuals. In a preferred embodiment, the donor individual can be a person different from the patient or recipient individual, and immune cells will be applied to the patient or recipient individual after the production (allogeneic treatment) as described herein. For example, the donor individual can be a healthy individual, and the healthy individual is a human leukocyte antigen (HLA) matched with a recipient individual suffering from cancer (before or after donation). In other embodiments, the donor individual may not be an HLA matched with the recipient individual. Preferably, the donor individual can be an infant (neonate), and for example, the donor cell can be obtained from an umbilical cord blood sample.

[0184] Suitable donor individuals are preferably free of infectious viruses (eg, HIV, HPV, CMV) and adventitious agents (eg, bacteria, mycoplasma), and are free of known genetic abnormalities.

[0185] In some embodiments, a peripheral blood cell population such as HPC for reprogramming can be isolated from a blood sample obtained from a donor individual, preferably an umbilical cord sample. Suitable methods for separating HPC and other peripheral blood cells are well known in the art and include, for example, magnetically activated cell sorting (see, for example, Gaudernack et al., 1986 Journal of Immunology Methods (J Immunol Methods) 90 179), fluorescence activated cell sorting (FACS: see, for example, Rheinherz et al., (1979) Proceedings of the National Academy of Sciences (PNAS) 76-4061) and cell panning (see, for example, Lum et al., (1982) Cellular Immunology 72-122). HPC can be identified in a blood cell sample by the expression of CD34. In other embodiments, a fibroblast population for reprogramming can be isolated from a skin biopsy after being dispersed using collagenase or trypsin and grown outward under suitable cell culture conditions.

[0186] Donor cells are usually reprogrammed into iPSCs by introducing reprogramming factors such as Oct4, Sox2 and Klf4 into cells. Reprogramming factors can be proteins or encoding nucleic acids and can be introduced into differentiated cells by any suitable technique (including plasmids, transposons or more preferably, by viral transfection or direct protein delivery). Other reprogramming factors, such as Klf genes, such as Klf-1, -2, -4 and -5; Myc genes, such as C-myc, L-myc and N-myc; Nanog; SV40 large T antigen; Lin28; and short hairpins (shRNA) targeting genes such as p53 can also be introduced into cells to increase induction efficiency. After the introduction of reprogramming factors, donor cells can be cultured. Cells expressing pluripotency markers can be separated and / or purified to produce iPSC colonies. Techniques for generating iPSCs are well known in the art (Yamanaka et al., Nature 2007;448:313-7; Yamanaka, 2007 Jun 7;1(1):39-49; Kim et al., Nature 2008 Jul 31;454(7204):646-50; Takahashi, Cell 2007 Nov 30;131(5):861-72. Park et al., Nature 2008 Jan 10;451(7175):141-6; Kimet et al., Cell Stem Cell 2009 Jun 5;4(6):472-6; Vallier, L. et al., Stem Cell 2009 Jun 5;4(6):472-6). Cells, 2009. 9999(999A): pN / A; Baghbaderani et al., 2016; Stem Cell Rev. 2016 Aug; 12(4): 394-420; Baghbaderani et al., (2015) Stem Cell Reports, 5(4), 647-659).

[0187] iPSCs can be cultured and maintained using conventional techniques (Vallier, L. et al., Developmental Biology (Dev.Biol.) 275, 403-421 (2004); Cowan, CA et al., New England Journal of Medicine (N.Engl.J.Med.) 350, 1353-1356 (2004), Joannides, A. et al., Stem Cells 24, 230-235 (2006), Klimanskaya, I. et al., Lancet (Lancet) 365, 1636-1641 (2005), Ludwig, TE et al., Nature Biotechnology 24, 185-187 (2006)). iPSCs used in the methods of the present invention can be grown under defined conditions or on feeder cells. For example, it can be conventionally grown at an appropriate density (e.g., 10 5 Up to 10 6 iPSCs are cultured in a culture dish on a feeder cell layer such as irradiated mouse embryonic fibroblasts (MEFs) or on a suitable substrate in a feeder-conditioned or defined iPSC maintenance medium (cells / 60 mm dish). iPSCs used in the methods of the present invention can be passaged enzymatically or mechanically. In some embodiments, iPSCs can be passaged in a culture dish such as mTeSR TM 1 or TeSR TM 2 (StemCell Technologies) or E8 flex (Life Thermo) medium, iPSC maintenance medium, in matrigel TM Or passage iPSCs on ECM proteins such as vitronectin.

[0188] IPSCs can be transfected with nucleic acids comprising heterologous expression cassettes so that the cassettes are integrated into the genome of the IPSCs. Suitable techniques are well known in the art. Transfection at the iPSC stage allows the isolation of monoclonal clones and the differentiation of homogenous cell populations.

[0189] Nucleic acid can be introduced into cells by any convenient technology. Suitable techniques for transporting heterologous expression cassettes to iPSC are well known in the art, and include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, gene editing (e.g., AAV-mediated gene editing) entering a specific location by gene editing, and transduction using retrovirus or other viruses (e.g., vaccinia virus or slow virus). In certain embodiments, CAS9 guide RNA ribonucleoprotein complexes (RNP) can be delivered by electroporation, and nucleic acid molecules (e.g., DNA molecules) such as targeting vectors encoding expression cassettes will be packaged as rAAV (serotype 6). Alternatively, RNA and DNA molecules encoding expression cassettes (e.g., ssDNA) can be co-delivered by electroporation.

[0190] By using CRISPR / Cas9 in combination to produce double-strand breaks and homology arms within nucleic acid molecules, targeting of integration sites in the genome of iPSCs is provided. Suitable integration sites include loci and safe harbor loci, and are described in more detail above. All clones can be screened to confirm integration at the correct site.

[0191] When heterologous nucleic acid is introduced or incorporated into iPSC, certain factors must be considered. The nucleic acid to be inserted should be assembled in a construct or vector containing an effective regulatory element, which will drive the transcription in T cells. For example, many known techniques and schemes for manipulating and converting nucleic acids such as preparing nucleic acid constructs, introducing DNA into cells, and gene expression are described in detail in "Protocols in Molecular Biology" Second Edition, Ausubel et al., edited by John Wiley & Sons, 1992. In certain embodiments, nucleic acid can be introduced into cells by gene editing. For example, DNA double-strand breaks (DSBs) at target sites can be induced by CRISPR / Cas9 systems, and the repair of DSBs can introduce heterologous nucleic acids into the cell genome at target sites, or nucleic acids can be introduced using rAAV vectors (AAV-mediated gene editing; Hirsch et al., 2014 "Molecular Biology Methods" 1114 291-307).

[0192] Also provided is an IPSC comprising a heterologous expression cassette integrated into its genome,

[0193] Wherein the expression cassette comprises:

[0194] (a) a coding sequence for producing a T cell receptor (TCR),

[0195] (b) a constitutive promoter operably linked to the coding sequence, and

[0196] (c) Targeting site.

[0197] The targeting site may be a 5' targeting site, and the cassette may further comprise a 3' targeting site.

[0198] Heterologous expression cassettes are described in more detail above.

[0199] iPSC can be differentiated and matured into immune cells such as T cells in a series of steps. The differentiation and maturation of the cell population in these steps are induced by culturing cells in a culture medium supplemented with a group of differentiation factors. The group of differentiation factors of each culture medium is preferably exhaustive, and the culture medium may lack other differentiation factors. In a preferred embodiment, the culture medium is a chemically defined culture medium. For example, as described below, the culture medium may be composed of a chemically defined nutrient medium, and the chemically defined nutrient medium is supplemented with an effective amount of one or more differentiation factors. The chemically defined nutrient medium may include a basal medium supplemented with one or more serum-free culture medium supplements.

[0200] Differentiation factor is to regulate, for example promote or inhibit the factor of the signal transduction pathway that mediates mammalian cell differentiation.Differentiation factor can include the growth factor, cytokine and small molecule that regulates one or more in activin / knot, FGF, Wnt or BMP or its signal transduction pathway.The example of differentiation factor includes activin / knot, FGF, BMP, retinoic acid, vascular endothelial growth factor (VEGF), stem cell factor (SCF), TGF beta ligand, GDF, LIF, interleukin, GSK-3 inhibitor and phosphatidylinositol 3-kinase (PI3K) inhibitor.

[0201] One or more differentiation factors used in the culture media described herein include TGFβ ligands, such as activin, fibroblast growth factor (FGF), bone morphogenetic protein (BMP), stem cell factor (SCF), vascular endothelial growth factor (VEGF), GSK-3 inhibitors (such as CHIR-99021), interleukins, and hormones such as IGF-1 and angiotensin II. Differentiation factors can be present in the culture media described herein in an amount effective to modulate signal transduction pathways in cells cultured in the culture media.

[0202] In some embodiments, the differentiation factors listed above or below can be replaced in the culture medium with factors that have the same effect (ie, stimulation or inhibition) on the same signaling pathway. Suitable factors are known in the art and include proteins, nucleic acids, antibodies, and small molecules.

[0203] By monitoring and / or detecting the expression of one or more cell markers in the differentiated cell colony, the degree of differentiation of the cell colony during each step can be determined.For example, it can be determined that the marker characteristic expression of the cell type with a higher degree of differentiation increases or the marker characteristic expression of the cell type with a lower degree of differentiation reduces.The expression of cell markers can be determined by any suitable technology, including immunocytochemistry, immunofluorescence, RT-PCR, immunoblotting, fluorescence-activated cell sorting (FACS) and enzymatic analysis.In a preferred embodiment, if the marker can be detected on the cell surface, it can be said that the cell expresses the marker.For example, the cell that does not express the marker as stated herein can show the active transcription and intracellular expression of the marker gene, but there may not be a marker of detectable level on the cell surface.

[0204] The partially differentiated cells (e.g., mesodermal cells, hemogenic endothelial (HE; i.e., hemogenic endothelial cells or HECs), HPCs, or T cell progenitors) populations produced by the steps in the methods described herein can be cultured, maintained, or expanded prior to the next differentiation step. The partially differentiated cells can be expanded by any convenient technique.

[0205] After each step, the partially differentiated cell colony produced by the steps may be free of or substantially free of other cell types. For example, after culturing in a culture medium, the colony may contain 60% or more, 70% or more, 80% or more, or 90% or more partially differentiated cells. Preferably, the cell colony is fully free of other cell types, and therefore purification is not required. If desired, the partially differentiated cell colony may be purified by any convenient technique such as MAC or FACS.

[0206] In the absence of feeder cells, cells can be cultured as a monolayer on a surface or substrate coated with an extracellular matrix protein such as fibronectin, laminin or collagen. Suitable techniques for cell culture are well known in the art (see, e.g., Basic Cell Culture Protocols, C. Helgason, Humana Press Inc. US (October 15, 2004) ISBN: 1588295451; Human Cell Culture Protocols (Methods in Molecular Medicine. S.) Humana Press Inc. US (December 9, 2004) ISBN: 1588292223; Culture of Animal Cells: A Manual of Basic Technique, R. Freshney, John Wiley & Sons (August 2, 2005) ISBN: 0471453293, Ho WY et al., Journal of Immunological Methods (2006) 310:40-52, Handbook of Stem Cells (2006) 311:40-53, Cells (ed. by R. Lanza) ISBN: 0124366430). Basic Cell Culture Protocols by J. Pollard and JM Walker (1997), Mammalian Cell Culture: Essential Techniques by A. Doyle and JB Griffiths (1997), Human Embryonic Stem Cells by A. Chiu and M. Rao (2003), Stem Cells: From Bench to Bedside by A. Bongso (2005), Human Stem Cell Manual: A Laboratory Guide by Peterson and Loring (2012) Academic Press and Human Embryonic Stem Cell Protocols by K. Turksen (2006).The culture medium and its components can be obtained from commercial sources (e.g., Gibco, Roche, Sigma, Europabioproducts, R&D Systems). Standard mammalian cell culture conditions can be used for the above culture steps, e.g., 37° C., 5% or 21% oxygen, 5% carbon dioxide. The culture medium is preferably changed every two days, and the cells are allowed to settle by gravity.

[0207] The cells may be cultured in a culture vessel. Suitable cell culture vessels are well known in the art and include culture plates, petri dishes, flasks, bioreactors and multi-well plates, for example, 6-well plates, 12-well plates or 96-well plates.

[0208] The culture vessel is preferably treated for tissue culture, for example by coating one or more surfaces of the vessel with an extracellular matrix protein such as fibronectin, laminin or collagen. The culture vessel can be treated for tissue culture using standard techniques (e.g., by incubation with a coating solution as described herein), or pre-treated culture vessels can be obtained from commercial suppliers.

[0209] iPSCs can be differentiated into immune cells using a multi-step process that includes:

[0210] (i) differentiating the iPSCs into mesoderm cells,

[0211] (ii) differentiating the mesodermal cells into hemogenic endothelial cells (HEC),

[0212] (iii) differentiating HECs into hematopoietic progenitor cell (HPC) populations,

[0213] (iv) differentiating the HPCs into immune cell progenitor cells; and

[0214] (v) maturing the progenitor immune cell population to generate an immune cell population expressing the heterologous expression cassette.

[0215] In the first stage, the iPSC colony can be differentiated into mesodermal cells. Mesodermal differentiation can be promoted, for example, by culturing the iPSC colony under suitable conditions, so that the iPSC is differentiated into mesodermal cells. For example, the iPSC cells can be cultured in the first mesoderm induction medium, the second mesoderm induction medium, and the third mesoderm induction medium in sequence to induce differentiation into mesoderm cells. In a preferred embodiment, the first mesoderm induction medium, the second mesoderm induction medium, and the third mesoderm induction medium are chemically defined culture media. For example, the first mesoderm induction medium can be composed of a chemically defined nutrient medium supplemented with an effective amount of activin, preferably activin A, e.g., 50 ng / ml activin A; the second mesoderm induction medium can be composed of a chemically defined nutrient medium supplemented with an effective amount of: activin, preferably activin A, e.g., 5 ng / ml activin A; BMP, preferably BMP4, e.g., 10 ng / ml BMP4; and FGF, preferably bFGF (FGF2), e.g., 5 ng / ml bFGF; and the third mesoderm induction medium can be composed of a chemically defined nutrient medium supplemented with an effective amount of: activin, preferably activin A, e.g., 5 ng / ml activin A; BMP, preferably BMP4, e.g., 10 ng / ml BMP4; FGF, preferably bFGF (FGF2), e.g., 5 ng / ml bFGF; and a GSK3 inhibitor, preferably CHIR-99021, e.g., 10 μM CHIR-99021.

[0216] In the second stage, mesodermal cells can be differentiated into hemogenic endothelial cells. Mesodermal cells can be differentiated into hemogenic endothelial (HE) cells by culturing a mesodermal cell population under suitable conditions that promote HE differentiation. For example, mesodermal cells can be cultured in a HE induction medium. In a preferred embodiment, the HE induction medium is a chemically defined medium. For example, the HE induction medium can be composed of a chemically defined nutrient medium supplemented with an effective amount of VEGF, for example, 15 ng / ml VEGF; and SCF, for example, 100 ng / ml SCF. Preferably, mesodermal cells are cultured in a HE induction medium consisting of a chemically defined nutrient medium and two differentiation factors, wherein the two differentiation factors are SCF and VEGF.

[0217] In the third stage, the hematopoietic endothelial cells can be differentiated into hematopoietic progenitor cells (HPCs). The hematopoietic endothelial (HE) cells can be differentiated into hematopoietic progenitor cells (HPCs) by culturing a population of HE cells under suitable conditions that promote hematopoietic differentiation. For example, HE cells can be cultured in a hematopoietic induction medium. In a preferred embodiment, the hematopoietic induction medium is a chemically defined medium. For example, the hematopoietic induction medium can be composed of a chemically defined nutrient medium supplemented with an effective amount of: VEGF, e.g., 15 ng / ml; SCF, e.g., 100 ng / ml; thrombopoietin (TPO), e.g., 30 ng / ml; Flt3 ligand (FLT3L), e.g., 25 ng / ml; IL-3, e.g., 25 ng / ml; IL-6, e.g., 10 ng / ml; IL-7, e.g., 10 ng / ml; IL-11, e.g., 5 ng / ml; IGF-1, e.g., 25 ng / ml; BMP, e.g., 10 ng / ml of BMP4; FGF, e.g., 5 ng / ml of bFGF; sonic hedgehog (SHH), e.g., 25 ng / ml; erythropoietin (EPO), e.g., 2 u / ml; angiotensin II, e.g., 10 μg / ml, and angiotensin II Type 1 receptor (AT1) antagonist, for example, losartan, 100 μM.

[0218] In the fourth stage, the HPCs can be differentiated into immune cell progenitors such as T cell progenitors. Hematopoietic progenitor cells (HPCs) can be differentiated into progenitor immune cells by culturing a population of HPCs under suitable conditions that promote lymphoid differentiation. For example, the hematopoietic progenitor cells can be cultured in a lymphoid expansion medium. In a preferred embodiment, the lymphoid expansion medium is a chemically defined medium. For example, the lymphoid expansion medium can consist of a chemically defined nutrient medium supplemented with an effective amount of the above-mentioned differentiation factors. Suitable lymphoid expansion mediums are well known in the art and include Stemspan TM SFEMII (Cat. No. 9605; Stem Cell Technologies, Inc., California) and Stemspan TM Lymphoid Expansion Supplement (Cat. No. 9915; Stem Cell Technologies, Inc., California).

[0219] In the fifth stage, the progenitor immune cells can be matured into TCRαβ+ immune cells such as TCRαβ+ T cells. The progenitor immune cells can be matured into TCRαβ+ immune cells by culturing a population of progenitor immune cells under suitable conditions that promote maturation. For example, the progenitor immune cells can be cultured in a maturation medium. In a preferred embodiment, the T cell maturation medium is a chemically defined medium. For example, the T cell maturation medium can be composed of a chemically defined nutrient medium supplemented with an effective amount of the above-mentioned differentiation factors. Suitable T cell maturation mediums are well known in the art and include Stemspan TM SFEMII (Cat. No. 9605; Stem Cell Technologies, Inc., California) and Stemspan TM T cell maturation supplement (Catalog No. 9930; California Stem Cell Technology, Inc.) and other culture media suitable for expanding PBMC and CD3+ cells, such as ExCellerate human T cell expansion medium (R&D Systems, Inc., USA). Other suitable T cell maturation culture media may include a basal culture medium supplemented with ITS, albumin and lipids as described elsewhere herein and further supplemented with an effective amount of the above-mentioned differentiation factors, such as IMDM.

[0220] In the sixth stage, TCRαβ+ immune cell colonies such as TCRαβ+T cells can be activated and / or amplified to produce or increase the ratio of single positive CD4+ immune cells, or more preferably to produce or increase the ratio of single positive CD8+ immune cells. Suitable methods for activating and amplifying immune cells such as T cells are well known in the art. For example, T cells can be exposed to T cell receptor (TCR) agonists under appropriate culture conditions. Suitable TCR agonists include ligands, such as peptides on Class I or Class II MHC molecules (MHC-peptide complexes) displayed on the surface of beads or antigen presenting cells such as dendritic cells, and soluble factors such as anti-TCR antibodies, such as anti-CD28 antibodies, and polymer MHC-peptide complexes such as MHC-peptide tetramers, pentamers or dextrorotatory bodies.

[0221] Suitable conditions and culture media for stages 1 to 6 are known in the art. Some preferred conditions and culture media are disclosed in WO2021 / 032836, WO2021 / 032855, WO2021 / 032851 and WO2021 / 032852, the contents of which (including conditions and culture media) are incorporated herein by reference.

[0222] Directed differentiation and maturation as described above produces an immune cell comprising a heterologous expression cassette. Also provided is an immune cell comprising a heterologous expression cassette integrated into its genome,

[0223] Wherein the expression cassette comprises:

[0224] (a) a coding sequence for producing a T cell receptor (TCR),

[0225] (b) a constitutive promoter operably linked to the coding sequence, and

[0226] (c) Targeting site.

[0227] The targeting site may be a 5' targeting site, and the cassette may further comprise a 3' targeting site.

[0228] Immune cells are described elsewhere herein. For example, the immune cell can be a TCRαβ+ immune cell, such as a TCRαβ+ T cell.

[0229] After generation, immune cells comprising heterologous expression cassettes can be stored or sensitized for therapy, such as adoptive cell therapy or adoptive immunotherapy. As described herein, immune cells are sensitized by introducing the expression construct encoding therapeutic TCR into the cell genome at the site of the heterologous expression cassette. For example, immune cells can be sensitized by replacing the heterologous expression cassette with an expression construct encoding therapeutic TCR as described herein. The sensitized immune cell colony expressing therapeutic TCR can be used as a medicine. For example, the immune cell colony expressing therapeutic TCR can be used for immunotherapy, such as adoptive cell therapy or adoptive immunotherapy.

[0230] Adoptive cell therapy or adoptive immunotherapy refers to the adoptive transfer of human immune cells such as T lymphocytes, which express antigens or peptides expressed on the patient's target cells and / or have specific TCRs for peptide MHC complexes expressed on the target cells. This can be used to treat a range of diseases, depending on the selected target, for example, tumor-specific antigens for the treatment of cancer. Adoptive cell therapy (ACT) involves removing a portion of the donor's cells, such as leukocytes. These cells are then used to produce iPSCs in vitro, and these iPSCs are transfected with heterologous expression cassettes, and used to efficiently produce immune cells. Immune cells can be amplified, washed, concentrated and / or then frozen, so that there is time to test, transport and store until the patient is ready to receive an infusion of immune cells. As described herein, immune cells are then sensitized by inserting an expression construct encoding a therapeutic TCR at the site of a heterologous expression cassette in the patient's body, wherein the therapeutic TCR has specificity for antigens or peptides expressed on target cells such as cancer cells, and / or has specificity for peptide MHC complexes on target cells such as cancer cells. The expression construct may replace the heterologous expression cassette. The nucleotide sequence encoding the therapeutic TCR may be derived from cancer-reactive immune cells such as tumor infiltrating lymphocytes obtained from a patient.

[0231] In some embodiments, as described herein, an immune cell population can be sensitized by inserting an expression construct population encoding a therapeutic TCR into an immune cell in a population at a site of a heterologous expression cassette, wherein the therapeutic TCR has specificity for different antigens or peptides expressed on target cells such as cancer cells, and / or has specificity for different peptide MHC complexes on target cells such as cancer cells of the patient. For example, a heterologous expression cassette can be substituted for an expression construct. The therapeutic TCR encoded by the expression construct population can be reactive with different tumor antigens of the patient. The nucleotide sequences encoding different therapeutic TCRs in the expression construct population can be derived from cancer reactive immune cells such as tumor infiltrating lymphocytes obtained from the patient. The immune cell population sensitized in this way can be reactive with a variety of different tumor antigens of the patient.

[0232] After generation and sensitization, the immune cell population expressing a therapeutic TCR or multiple therapeutic TCRs produced as described herein can be blended with other reagents such as buffers, carriers, diluents, preservatives and / or pharmaceutically acceptable excipients. Suitable reagents are described in more detail below. The methods described herein can include blending the immune cell population with a pharmaceutically acceptable excipient.

[0233] Pharmaceutical compositions suitable for administration (e.g., by infusion) include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions, which may contain antioxidants, buffers, preservatives, stabilizers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. Examples of suitable isotonic vehicles for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Suitable vehicles can be found in standard pharmaceutical literature, such as Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

[0234] In some preferred embodiments, the immune cells can be formulated into a pharmaceutical composition suitable for intravenous infusion into an individual.

[0235] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues of subjects (e.g., humans) without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other components of the formulation.

[0236] Other aspects of the invention provide the use of a population of immune cells expressing a therapeutic TCR or multiple therapeutic TCRs produced as described herein for the preparation of a medicament for treating cancer; a population of immune cells expressing a therapeutic TCR or multiple therapeutic TCRs produced as described herein for the treatment of cancer; and a method of treating cancer comprising administering a population of immune cells expressing a therapeutic TCR or multiple therapeutic TCRs produced as described herein to an individual in need thereof.

[0237] The immune cell population may be allogeneic, ie, the immune cells were originally obtained from an individual different from the individual to which they are subsequently administered (ie, the donor individual and the recipient individual are different). Allogeneic refers to a transplant derived from a different animal of the same species.

[0238] The donor and recipient individuals can be HLA matched to avoid GVHD and other undesirable immune effects, such as rejection. Alternatively, the donor and recipient individuals may not be HLA matched, or the HLA genes in the cells from the donor individual may be modified, for example by gene editing, to remove any HLA mismatch with the recipient.

[0239] A suitable population of immune cells for administration to a recipient individual can be produced by a method comprising providing an initial population of cells, preferably T cells, obtained from a donor individual, reprogramming the cells into iPSCs, transfecting the iPSCs with a heterologous expression cassette, differentiating the iPSCs into immune cells, and sensitizing the immune cells by: replacing the heterologous expression cassette in the recipient individual with an expression construct encoding a therapeutic TCR that specifically binds to an antigen or peptide thereof presented by a cancer cell and / or by a cancer cell, optionally in complex with an MHC; or replacing the heterologous expression cassette in the immune cells in the recipient individual with a population of expression constructs, each expression construct encoding a therapeutic TCR that specifically binds to a different antigen or peptide thereof presented by a cancer cell, optionally in complex with an MHC.

[0240] Following administration of immune cells expressing a therapeutic TCR or multiple therapeutic TCRs, the recipient individual may exhibit a cell-mediated immune response against the recipient individual's cancer cells. This may have a beneficial effect on the individual's cancer condition.

[0241]

[00136] As used herein, the terms "cancer," "neoplasm," and "tumor," and in the singular or plural, refer to cells that have undergone a malignant transformation that renders them pathological to the host organism.

[0242] Primary cancer cells can be easily distinguished from non-cancerous cells by mature techniques, particularly histological examination. Cancer cells include not only primary cancer cells, but also any cells derived from cancer cell ancestors. This includes metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that usually manifests as a solid tumor, a "clinically detectable" tumor is a tumor that is detectable based on tumor mass; for example, by procedures such as computed tomography (CT) scans, magnetic resonance imaging (MRI), X-rays, ultrasound, or physical examination palpation, and / or due to the expression of one or more cancer-specific antigens in a sample that can be obtained from a patient.

[0243] Cancer conditions can be characterized by the abnormal proliferation of malignant cancer cells and can include: leukemias such as AML, CML, ALL and CLL; lymphomas such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma; and solid cancers such as sarcomas, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, stomach cancer, testicular cancer, gallbladder and bile duct cancer, thyroid cancer, thymus cancer, bone cancer and brain cancer, and cancer of unknown primary (CUP).

[0244] Cancer cells in an individual may be immunologically different from normal somatic cells in the individual (i.e., a cancer tumor may be immunogenic). For example, a cancer cell may be able to elicit a systemic immune response in an individual to one or more antigens expressed by the cancer cell. The tumor antigen that elicits the immune response may be specific to the cancer cell, or may be shared by one or more normal cells in the individual.

[0245] Cancer cells of an individual suitable for treatment as described herein may express the antigen and / or may be of the correct HLA type to bind to the αβ TCR expressed by a T cell.

[0246] The individual suitable for treatment as described above can be a mammal. In a preferred embodiment, the individual is a human. In other preferred embodiments, non-human mammals can be used, particularly mammals (e.g., murines, primates, porcines, canines or rabbits) that are routinely used as models for demonstrating therapeutic efficacy in humans.

[0247] In some embodiments, after initial cancer treatment, an individual may have minimal residual disease (MRD).

[0248] An individual with cancer may exhibit at least one identifiable sign, symptom, or laboratory finding sufficient to make a diagnosis of cancer according to clinical criteria known in the art. Examples of such clinical criteria can be found in medical textbooks, such as Harrison's Principles of Internal Medicine, 15th edition, edited by Fauci AS et al., McGraw-Hill, New York, 2001. In some instances, diagnosis of cancer in an individual may include identifying a specific cell type (e.g., cancer cell) in a body fluid or tissue sample obtained from the individual.

[0249] An anti-tumor effect is a biological effect that can be manifested as a reduction in tumor growth rate, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer pathology. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells (particularly T cells) produced according to the methods of the invention, and the antibodies described herein to prevent tumorigenesis in the first place.

[0250] Treatment can be any treatment and / or therapy, whether for humans or animals (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, such as inhibiting or delaying the progression of a condition, and includes a reduction in the rate of progression, arrest of the rate of progression, amelioration of a condition, cure or alleviation of a condition (whether partial or total), prevention, delay, alleviation or arrest of one or more symptoms and / or signs of a condition, or prolonging the survival of a subject or patient beyond what would be expected in the absence of treatment.

[0251] Treatment can also be preventive (i.e., prophylactic). For example, an individual susceptible to cancer or at risk of developing or recurring cancer can be treated as described herein. Such treatment can prevent or delay the onset or recurrence of cancer in an individual.

[0252] Specifically, treatment can include inhibiting cancer growth, including complete remission of cancer and / or inhibiting cancer metastasis. Cancer growth generally refers to any one of many indicators indicating that cancer changes to a more serious form. Therefore, indicators for measuring cancer growth inhibition include a reduction in cancer cell survival, a reduction in tumor volume or morphology (e.g., as determined using computed tomography (CT), ultrasound scanning or other imaging methods), delayed tumor growth, destruction of tumor vasculature, improved performance in delayed hypersensitivity skin tests, an increase in T cell activity and a reduction in tumor-specific antigen levels. Administration of modified immune cells as described herein can improve the ability of an individual to resist cancer growth, specifically the growth of cancer already present in a subject, and / or reduce the tendency of cancer growth in an individual.

[0253] Immune cells or pharmaceutical compositions comprising immune cells can be administered to a subject by any convenient route of administration (e.g., by infusion), whether systemic / peripheral or at the site of desired action, including but not limited to parenteral. Infusion involves administering T cells in a suitable composition by a needle or catheter. Typically, T cells are infused intravenously or subcutaneously, although T cells can be infused by other non-oral routes, such as intramuscular injection and epidural routes. Suitable infusion techniques are known in the art and are typically used in therapy (see, e.g., Rosenberg et al., New England Journal of Medicine (NewEng.J.of Med.) 319:1676, 1988).

[0254] Generally, the number of cells administered is about 10 per kg body weight. 5 to about 10 10 cells per individual, such as about 1, 2, 3, 4, 5, 6, 7, 8 or 9, x10 5 、x10 6 、x10 7 、x10 8 、x10 9 or x10 10 Any of the following types of cells, usually 2x10 per individual 8 Up to 2x10 10Cells, usually within 30 minutes, are treated repeatedly as needed, for example, at intervals of several days to weeks. It should be understood that the appropriate dosage of TCRαβ+T cells and compositions comprising immune cells may vary from patient to patient. Determining the optimal dose will generally involve balancing the therapeutic benefit level of the treatment of the present invention with any risk or harmful side effects. The selected dosage level will depend on a variety of factors, including but not limited to the activity of specific cells, cytokine release syndrome (CRS), route of administration, time of administration, rate of cell loss or inactivation, duration of treatment, other drugs, compounds and / or materials used in combination, and the patient's age, sex, weight, condition, overall health and previous medical history. The amount of cells and route of administration will ultimately be determined by the doctor, although the dosage will generally reach a local concentration that achieves the desired effect at the site of action without causing substantial harmful or harmful side effects.

[0255] Although immune cells can be administered alone, in some cases, immune cells can be administered in combination with target antigens, APCs displaying target antigens, CD3 / CD28 beads, IL-2, IL7 and / or IL15 to promote in vivo expansion of immune cell populations. Combined administration can be performed by separate, simultaneous or sequential administration of the combined components.

[0256] Immune cell populations can be administered in combination with one or more other therapies, such as cytokines, e.g., IL-2, CD4+CD8+ chemotherapy, radiation, and immuno-oncology agents, including checkpoint inhibitors, such as anti-B7-H3, anti-B7-H4, anti-TIM3, anti-KIR, anti-LAG3, anti-PD-1, anti-PD-L1, and anti-CTLA4 antibodies. Combined administration can be performed by separate, simultaneous, or sequential administration of the combination components.

[0257] The one or more other therapies may be administered by any convenient means, preferably at a site separate from the site of administration of the immune cells.

[0258] Administration of immune cells can be carried out in a single dose, continuously or intermittently (e.g., divided doses at appropriate intervals) throughout the course of treatment. Methods for determining the most effective mode of administration and dosage are well known to those skilled in the art and will vary with the composition used for therapy, the purpose of therapy, the target cells to be treated, and the subject to be treated. Single or multiple administrations can be carried out by selecting the dosage level and mode by the treating physician. Preferably, immune cells are administered in a single transfusion, such as any of 500 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, 10 billion, 11 billion, 12 billion, 13 billion, 14 billion, 15 billion T cells, such as at least 1x10 9 cells.

[0259] Other aspects and embodiments of the present invention provide aspects and embodiments described above in which the term "comprising" is replaced by the term "consisting of" and aspects and embodiments described above in which the term "comprising" is replaced by the term "consisting essentially of.

[0260] It should be understood that the present application discloses all combinations of any of the above aspects and embodiments described above, unless the context otherwise requires. Similarly, unless the context otherwise requires, the present application discloses all combinations of individual preferred features and / or optional features or with any of the other aspects.

[0261] Modifications to the above-described embodiments, additional embodiments, and revisions thereof will be apparent to those skilled in the art from reading this disclosure and, thus, are intended to be within the scope of the present invention.

[0262] All documents and sequence database entries cited in this specification are herein incorporated by reference in their entirety for all purposes.

[0263] As used herein, "and / or" should be understood as explicitly disclosing each of the two specified features or components with or without the other. For example, "A and / or B" will be considered to explicitly disclose each of (i) A, (ii) B, and (iii) A and B, just as if each were listed individually herein.

[0264] experiment

[0265] iPSC cell culture

[0266] Knock-in of TCR landing pad constructs ( Figure 7 and Figure 8 ) was performed in the iPSC line GR1.1 (Baghbaderani et al. 2015; supra). For editing experiments, the GR1.1 iPSC line was maintained in a complete mTeSR coated with Matriclone (0.25 μg / cm2) (Solemtim). TMPlus medium (Stem Cell Technologies). GR1.1 iPSC line cells were passaged once every 4-5 days with Versene (ThermoFisher) or Accutase (Stem Cell Technologies). iPSC cultures were maintained in a humid 37°C, 5% O2, 5% CO2 incubator. The expression of pluripotency markers (POU5F1, NANOG, TRA-160 and SOX2) and the absence of differentiation marker SSEA-1 were routinely monitored by FACS analysis. Prior to differentiation, the edited iPSC clones were adapted for growth maintenance on Synthemax Matrrix (Corning). All other culture conditions were identical.

[0267] Generation of rAAV targeting vectors.

[0268] AAV targeting constructs were generated by Gibson assembly ( Figure 7 and 8 ). The homology arm regions were PCR amplified from genomic DNA, and the remaining components of the targeting vector were synthesized synthetically. rAAV vector (serotype 6) was generated by transiently transfecting HEK293T cells with the targeting vector and pDP6 packaging plasmid (Plasmid Factory). HEK293T was transfected with a plasmid:PEI ratio of 1 μg plasmid:1 μl PEI. rAAV was purified using iodixanol gradient ultracentrifugation according to standard protocols (Strobel et al., (2015) Hum Gen Ther Methods 26(4)147-157).

[0269] Guide RNA sequence

[0270] PTPRC exon 33 was targeted using the guide RNA GCAAGTCCAGCTTTAAATCA (SEQ ID NO: 9). (Chr1 198756152 to 198756171 (human GRCh38 - Ensembl 104 version - May 2021), PPP1r12C intron 1 was targeted with guide RNA GTCCCCTCCACCCCACAGTG (SEQ ID NO: 7) (Chr19: 55,115,770-55,115,790 human GRCh38 (Ensembl 104 version - May 2021). TRAC exon 1 was targeted with guide RNA AGAGTCTCTCAGCTGGTACA (SEQ ID NO: 6) (Chr1422547530 to 22547549 human GRCh38 - Ensembl 104 version - May 2021). Guide RNA sequences were synthesized by IDT.

[0271] Preparation of ribonucleoprotein (RNP) complexes

[0272] The crRNA and tracrRNA were ligated by initial denaturation at 95° C. for 5 minutes before cooling to room temperature. Equimolar amounts of ligated crRNA / tracrRNA duplexes and Cas9 protein (IDT) were incubated at room temperature for 15 minutes to generate 10 μM ribonucleoprotein (RNP) complexes.

[0273] Knock-in of ADB796 landing pad - targeting PTPRC exon 33 or PPP1R12C intron 1

[0274] By using a 16-well Nucleocuvette TM 4D-Nucleofector TM Nucleofection was performed to introduce RNP complexes targeting PTPRC exon 33 or PPP1R12C intron 1 into iPSC cells. 200x103GR1.1 were resuspended in buffer P3 (Lonza P3 Primary Cell 4D-Nucleofector TM ) (10x106 / ml). 3 μl of RNP complex (10 μM) was added to 20 μl of cell suspension. Nucleofection was performed using program CA-137. Immediately after nucleofection, cells were plated on plates supplemented with 1x CloneR TM(Stem Cell Technology Company) in complete mTESR Plus. AAV transduction (2x103 vector genomes / cell) was performed 6-8 hours after cell inoculation. The edited GR1.1 cells were then cultured in complete mTESR Plus. Before isolating iPSC clones derived from single cells and genotyping the edited clones, the cells were expanded for one passaging. Single cells were inoculated into 96-well plates using a VIPS instrument from Solentim and amplified for 10-14 days. Using primers corresponding to genomic DNA outside the homology arm region and within the TCR transgene, PCR genotyping was performed on the edited clones according to standard protocols. In addition, TLA analysis (Cergentis Company (Cergentis)) was used to confirm that the ADB796TCR landing pad was integrated into the desired genomic location (PTPRC exon 33 or PPP1R12C (intron 1)).

[0275] Exchange MAGE-A10 TCR ADB796 to MAGE-A4 / B2 TCR ADB959

[0276] Excise the ADB796 TCR landing pad using RNP containing the TGTACCAGCTGAGAGACTCT guide RNA. Differentiate PTPRCWT / ADB796 landing pad or PPP1R12CWT / ADB796 landing pad iPSC cells into iT cells. Collect CD4 / CD8 double positive iT cells at the end of differentiation (stage 5). Use a 16-well Nucleocuvette TM Bars, using P2 primary cells 4D-Nucleofector X kit S TM Perform nuclear transfection. 6 iT cells were resuspended in 20 μl P2. 3 μl RNP complex (10 μM) was added to 20 μl cell suspension. Nucleofection was performed using program EH100. AAV transduction (5x10 3 Vector genomes / cell). Cells were cultured for 72 hours before phenotyped by FACS. The expression of ADB796 and ADB959 in cells was analyzed by FACS by staining with anti-Vβ13.2 (specific for ADB796), anti-TCR Vα24 (specific for ADB959).

[0277] Landing Pad Strategy 1 Design and generation of TCR A2M10 placeholder construct (ADB00794_001).

[0278] The rAAV repair template encoding the recombinant AAV production vector was designed to allow constitutive expression of the A2M10 TCR from the EF-1a promoter. The expression cassette is flanked by a 41 bp sequence present in human B2M. The placeholder cassette consists of 6 elements, as shown in Figure 12:

[0279] o Right homology arm, required for homology-directed repair (HDR)-based integration of the cassette into the PPP1R12C (AAVS1) locus (present in humans, Chr19:55115773-55115274GRCh38.p14).

[0280] o The B2M target site present in human B2M (Chr15:44715435-44715475, GRCh38.p14) that served as a targetable DNA sequence to replace the placeholder TCR with the exchange TCR (A2M4).

[0281] ○EF-1α promoter.

[0282] ○A2M10 TCR sequence (Border et al., Oncoimmunology, 2018)

[0283] ○SV40 polyadenylation signal

[0284] o Left homology arm, required for homology-directed repair (HDR)-based integration of the cassette into the PPP1R12C (AAVS1) locus (present in human PPP1R12C, Chr19:55116272-55115774, GRCh38.p14).

[0285] This construct was designed to be cloned into the rAAV production backbone (Agilent pAAV_MCS) using Gibson cloning.

[0286] Landing Pad Strategy 1 Generation of TCR A2M10 Placeholder Construct: ADB00794_001

[0287] The A2M10 expression cassette was synthesized by Twist Biosciences and inserted into the pTwist-puro backbone. Next, the A2M10 expression cassette was PCR amplified using primers containing the 41 bp B2M (Chr15: 44715435-44715475, GRCh38.p14) target sequence (SEQ ID NOs: 42 and 43).

[0288] FWD and REV primers of SEQ ID NOs 44 and 45 were used to amplify the left homology arm (LHA) (present in humans, Chr19: 55115774-55116274GRCh38.p14) required from genomic DNA isolated from GR1.1 iPSC (Baghbaderani et al., Stem Cell Reports, 2015) cells.

[0289] The right homology arm (RHA) was amplified from genomic DNA of GR1.1 iPSC cells using FWD and REV primers of SEQ ID NOs 46 and 47 (present in humans, Chr19: 55115773-55115274GRCh38.p14):

[0290] All PCRs were performed with Q5 DNA polymerase (NEB, M0491L) according to standard protocols. The PCR product - the A2M10 expression cassette flanked by the B2M target sequence, RHA and LHA was purified by gel extraction using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, 740609.50) according to the manufacturer's instructions and purified using Gibson The clones were assembled into a NotI-digested adeno-associated virus vector backbone (Agilent pAAV-MCS) using an equimolar ratio of the DNA fragments using a cloning kit (NEB, E5510S). Clones were screened by restriction enzyme digestion and the sequences were verified by Sanger sequencing.

[0291] Landing Pad Strategy 1 Generation of TCR A2M4 Exchange Constructs: ADB01032_026

[0292] The A2M4TCR_BGHpolyA expression plasmid was synthesized by GeneART. A2M4TCR_BGHpolyA (Sanderson et al., Tumor Immunology, 2019) was PCR amplified using FWD and REV primers of SEQ ID NOs 48 and 49.

[0293] The left homology arm (LHA) containing a 500 bp sequence (present in human PPP1R12C, Chr19:55116273-55115793GRCh38.p14) was amplified from ADB00794_001 using FWD and REV primers of SEQ ID NOs 50 and 51.

[0294] The right homology arm (RHA) containing a 501 bp sequence (present in human PPP1R12C, Chr19:55115775-55115274GRCh38.p14) was amplified from ADB00794_001 using FWD and REV primers of SEQ ID NOs 52 and 53.

[0295] All PCRs were performed with Q5 DNA polymerase (NEB, M0491L) according to standard protocols. PCR products - A2M4TCR_BGH polyA, RHA and LHA and EF-1α promoter were purified by gel extraction using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, 740609.50) and purified using Gibson The clones were assembled into a NotI-digested adeno-associated virus vector backbone (pAAV-MCS) using an equimolar ratio of DNA fragments using a cloning kit (NEB, E5510S). Clones were screened by restriction enzyme digestion and verified by Sanger sequencing. The EF-1α promoter was amplified from ADB00794-001 using a forward primer (GGCTCCGGTGCCCGTCAGTGGGC) and a reverse primer (GGTGGCGGCAAGCTTGGCAGCGGC).

[0296] iPSC cell culture

[0297] TCR landing pad structure ( Figure 2 ) knock-in was performed in the iPSC line GR1.1 (Baghbaderani et al., 2015). For editing experiments, the GR1.1 iPSC line was maintained in complete mTeSR coated with vitronectin (0.5 μg / cm2) (Gibco, A14700). TM Plus medium (Stem Cell Technologies, 100-0276) was used to treat tissue culture. GR1.1 iPSC line cells were passaged every 4-5 days with Versene (Thermo Fisher Scientific, 15040066) or Accutase (Stem Cell Technologies, 07920). iPSC cultures were maintained in a humidified 37°C, 5% O2, 5% CO2 incubator.

[0298] Generation of A2M10 placeholder TCR knock-in iPSC cells.

[0299] The guide RNA targeting intron 1 PPP1R12C locus with the sequence GTCCCCTCCACCCCACAGTG (SEQ ID NO:7; Chr19: 55,115,770-55,115,790 human GRCh38 - Ensembl version 104 - May 2021) was synthesized as a single guide RNA by Synthego. The ADB00794_001 repair template was packaged into AAV6 and purified by Virovek. The placeholder cassette was knocked into GR1.1 iPSC cells using CRISPR-Cas9 using purified AAV6-ADB00794_001 virus. In brief, 250,000 cells were electroporated with 62 pmoles of high-fidelity SpyFi Cas9 (Aldevron, 9214-0.25MG) with a 1.2 molar ratio of guide RNA to 4D-Nucleofector TM The PPP12R1C locus was targeted using the CA-137 protocol on the system. Using a 16-well Nucleocuvette TM Bars, using P3 primary cells 4D-Nucleofector X kit S TM After electroporation, transfer the cell suspension to a 500 μl complete mTESR TM Plus 24-well plates supplemented with 1x CloneR TM 2 (Stem Cell Technologies, Inc., 100-0691) and 1.25x10 9 AAV6-ADB01032_026vg. The cells were then cultured for two weeks and single cells were seeded into 96-well plates using the Solentim validated in situ plating (VIPS) platform. Cells were screened for targeted transgene integration using junction PCR (Geisinger, 2016, Nucleic Acids Research). Junction PCR primers of SEQ ID NOs 54-57 were used.

[0300] Integration was confirmed at both the 5' and 3' ends to maximize confidence in the correct gene editing outcome. The allele frequency of the integrated landing pad cassette was confirmed by amplicon PCR followed by agarose gel electrophoresis.

[0301] rAAV6-A2M4 was generated by exchanging the repair template.

[0302] rAAV was produced by transient transfection of suspended HEK293T using a dual plasmid system. 2mM sodium butyrate was included 24 hours after transfection. Cells were collected by centrifugation (350g, 5 minutes) 48 hours after transfection, washed with PBS, and resuspended in 5mM Tris pH8.5, 150mM NaCl (18ml / 250ml original culture volume). Resuspended cells were lysed by freeze-thaw (frozen on dry ice and thawed in a 37°C water bath). Cell lysates were treated with omnipotent nuclease (250U / ml, MgCl2 was added to a concentration of 2mM) at 37°C for 1 hour. The lysate treated with omnipotent nuclease was clarified by centrifugation (4000xg, 30 minutes), and the supernatant was filtered 0.45um before being purified by chromatography on (). The clarified lysate was loaded onto a POROS Capture Select AAVX 1 ml column at a flow rate of 0.5 ml / min, washed with high salt buffer (10 mM Tris pH 8, 1 M NaCl), and eluted with low pH glycine buffer (50 mM glycine pH 2.7, 500 mM NaCl). The eluted AAV was neutralized by adding Tris pH 8 to a concentration of 80 mM and analyzed by SDS-PAGE and dPCR.

[0303] Generation of progenitor T cells carrying the A2M10 placeholder.

[0304] iPSC clones were differentiated into CD34+ hematopoietic progenitor stem cells and then into CD3+ iT cell progenitors according to an in-house protocol. Expression of the A2M10 placeholder TCR was confirmed by flow cytometry ( Fig.13 ).

[0305] The A2M10 placeholder TCR in early T cell progenitors is exchanged for A2M4.

[0306] In cloning 15F2_AAVS1 from iPSC - / A2M10LP and 16D5_AAVS1 A2M10LP / A2M10LP A2M10 TCR was exchanged for A2M4 TCR in iT cells that were differentiated and at different differentiation stages. 15F2_AAVS1 was expressed by Cas9-guide RNA ribonucleoprotein (RNP). - / A2M10LP 3.5x10 5iT progenitor cells were electroporated (SEQ ID NO: 63: ucacgucauccagcagagaa) and transduced with rAAV6-ADB01032_026 immediately after electroporation (as described in Figure 12). The culture medium was changed 48 hours after transduction, and flow cytometry analysis was performed 24 hours later (Figures 14, 15). The list of antibodies used can be found in Table 2. DNA PK inhibitors (M3814, S8586, Selleckchem) were used to improve HDR editing results (Riesenberg et al., 2019 "Nucleic Acids Research", Fu et al., 2021 "Nucleic Acids Research"). A2M10 placeholder TCR exchange in the independent cell line 16D5_AAVS1 A2M10LP / A2M10LP This was reproduced in the GR1.1 line (Figures 16 and 17).

[0307] The A2M10 placeholder TCR in late T cell progenitors is exchanged for A2M4.

[0308] 1x10 6 Late 15F2_AAVS1 - / A2M10LP T cell progenitors were electroporated and treated with ImmunoCult TM After activation with human CD3 / CD28 T cells (STEMCELL TECHNOLOGIES, 10971), cells were transduced with AAV6-A2M4 on day 38. The cells were transduced using a 96-well Nucleocuvette using the DZ100 program. TM Plate, using P3 primary cells 4D-Nucleofector X kit TM Electroporation was performed. The medium was changed 48 hours after transduction and flow cytometry analysis was performed 48 hours later (Figures 18, 19). The list of antibodies used can be found in Table 2.

[0309]

[0310]

[0311] Table 1 - Safe Harbor Loci

[0312]

[0313] Table 2

[0314] sequence

[0315] GSGATNFSLL KQAGDVEENP GP

[0316] SEQ ID NO:1-P2A cleavage sequence

[0317] GGAAGCGGAGCT ACTAACTTCA GCCTGCTGAA GCAGCTGGAGACGTGGAGG AGAACCCTGGGCCT

[0318] SEQ ID NO:2—Nucleotide sequence encoding P2A peptide

[0319] GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG

[0320] SEQ ID NO:3—Ef1a short promoter

[0321] CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

[0322] SEQ ID NO:4—Bovine growth hormone PolyA signal

[0323] TCTCTCAGCTGGTACACGGC

[0324] SEQ ID NO:5-TRAC exon 1 Chr14 22547526 to 22547545 (-) (human GRCh38 - Ensembl 104 version - May 2021)

[0325] AGAGTCTCTCAGCTGGTACA

[0326] SEQ ID NO:6-TRAC exon 1 Chr14 22547530 to 22547549 (-) (human GRCh38-Ensembl104 version-May 2021)

[0327] GTCCCCTCCACCCCACAGTG

[0328] SEQ ID NO:7-PPP1R12C Intron 1Chr19:55,115,770-55,115,790 (human GRCh38 (Ensembl 104 version - May 2021)

[0329] TCACGTCATCCAGCAGAGAA

[0330] SEQ ID NO:8-B2M exon 2CHr15 44715446 to 44715465 (human GRCh38 - Ensembl version 104 - May 2021)

[0331] GCAAGTCCAGCTTTAAATCA

[0332] SEQ ID NO:9-PTPRCChr 1 198756152 to 198756171 (+) (human GRCh38 - Ensembl version 104 - May 2021)

[0333] AACTTGTTTATTGCAGCTTAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTA

[0334] SEQ ID NO:10—SV40 PolyA sequence

[0335] CGGGCCAAGAGAAGCGGATCCGGC

[0336] SEQ ID NO: 11 - Nucleotide sequence encoding the furin cleavage site and SG linker

[0337] RAKRSGS

[0338] SEQ ID NO: 12—Peptide sequence encoding the furin cleavage site and SG linker

[0339] CAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGT

[0340] SEQ ID NO: 13 - Truncated TRAC domain - Nucleotide sequence

[0341] ATGTCTCTGGGCCTGCTGTGCTGTGGCGTGTTCTCCCTGCTGTGGGCCGGACCTGTGAATGCCGGCGTGACCCAGACCCCCAAGTTCCGGGTGCTGAAAACCGGCCAGAGCATGACACTGCTGTGCGCCCAGGACATGAACCACGACTACATGTATTGGTACAGACAGGACCCCGGCATGGGCCTGCGGCTGATCCACTATTCTGTGGGCGAGGGCACCACCGCCAAGGGCGAAGTGCCTGATGGCTACAACGTGTCCCGGCTGAAGAAGCAGAACTTCCTGCTGGGCCTGGAAAGCGCCGCTCCTAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCTTCACCGACACCCAGTACTTCGGCCCTGGCACCAGACTGACCGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCCTCTGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTGGTCTGCCTGGCCACCGGCTTCTACCCCGACCACGTGGAACTGTCTTGGTGGGTGAACGGCAAAGAGGTGCACAGCGGCGTCAGCACCGACCCTCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCAGACTGCGGGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCTGTGACCCAGATCGTGTCTGCCGAAGCATGGGGGCGCGCCGATTGCGGCTTCACAAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCTCTGGTGCTGATGGCCATGGTGAAACGGAAGGACAGCCGGGGC

[0342] SEQ ID NO:14 - Nucleotide sequence of the c796 TCRβ chain of MAGE - A10

[0343] MSLGLLCCGVFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCAQDMNHDYMYWYRQDPGMGLRLIHYSVGEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSFTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0344] SEQ ID NO:15 - Amino acid sequence of the TCRβ chain of MAGE - A10 c796

[0345] ATGATGAAGTCCCTGCGGGTGCTGCTGGTCATCCTGTGGCTGCAGCTGTCCTGGGTCTGGTCCCAGCAGAAAGAGGTGGAGCAGAACAGCGGCCCTCTGAGCGTGCCCGAGGGCGCTATCGCCAGCCTGAACTGCACCTACAGCGACAGAGGCAGCCAGAGCTTCTTCTGGTACAGACAGTACAGCGGCAAGAGCCCCGAGCTGATCATGAGCATCTACAGCAACGGCGACAAAGAGGACGGCCGGTTCACCGCCCAGCTGAACAAGGCCAGCCAGTACGTGTCCCTGCTGATCCGGGACAGCCAGCCCAGCGACAGCGCCACCTACCTGTGCGCCGTGAGAGGCACAGGCAGAAGGGCCCTGACATTTGGCAGCGGCACCAGACTGCAGGTGCAGCCCAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC

[0346] SEQ ID NO:16 - Nucleotide sequence of the c796 TCRα chain of MAGE - A10

[0347] MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVRGTGRRALTFGSGTRLQVQPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0348] SEQ ID NO:17 - Amino acid sequence of the c796 TCRα chain of MAGE - A10

[0349] 。

[0350] SEQ ID NO: 18 - PTPRC exon 33 targeting vector - left homology arm (Chr1: 198755130-198756201 Human GRCh38 - Ensembl version 104 - May 2021)

[0351] 。

[0352] SEQ ID NO:19 - PTPRC exon 33 targeting vector - right homology arm (chromosome 1: 198,756,132-198,757,230 human GRCh38 - Ensembl version 104 - May 2021)

[0353] GCTCCCATAGCTCAGTCTGGTCTATCTGCCTGGCCCTGGCCATTGTCACTTTGCGCTGCCCTCCTCTCGCCCCCGAGTGCCCTTGCTGTGCCGCCGGAACTCTGCCCTCTAACGCTGCCGTCTCTCTCCTGAGTCCGGACCACTTTGAGCTCTACTGGCTTCTGCGCCGCCTCTGGCCCACTGTTTCCCCTTCCCAGGCAGGTCCTGCTTTCTCTGACCTGCATTCTCTCCCCTGGGCCTGTGCCGCTTTCTGTCTGCAGCTTGTGGCCTGGGTCACCTCTACGGCTGGCCCAGATCCTTCCCTGCCGCCTCCTTCAGGTTCCGTCTTCCTCCACTCCCTCTTCCCCTTGCTCTCTGCTGTGTTGCTGCCCAAGGATGCTCTTTCCGGAGCACTTCCTTCTCGGCGCTGCACCACGTGATGTCCTCTGAGCGGATCCTCCCCGTGTCTGGGTCCTCTCCGGGCATCTCTCCTCCCTCACCCAACCCCATGCCGTCTTCACTCGCTGGGTTCCCTTTTCCTTCTCCTTCTGGGGCCTGTGCCATCTCTCGTTTCTTAGGATGGCCTTCTCCGACGGATGTCTCCCTTGCGTCCCGCCTCCCCTTCTTGTAGGCCTGCATCATCACCGTTTTTCTGGACAACCCCAAAGTACCCCGTCTCCCTGGCTTTAGCCACCTCTCCATCCTCTTGCTTTCTTTGCCTGGACACCCCGTTCTCCTGTGGATTCGGGTCACCTCTCACTCCTTTCATTTGGGCAGCTCCCCTACCCCCCTTACCTCTCTAGTCTGTGCTAGCTCTTCCAGCCCCCTGTCATGGCATCTTCCAGGGGTCCGAGAGCTCAGCTAGTCTTCTTCCTCCAACCCGGGCCCCTATGTCCACTTCAGGACAGCATGTTTGCTGCCTCCAGGGATCCTGTGTCCCCGAGCTGGGACCACCTTATATTCCCAGGGCCGGTTAATGTGGCTCTGGTTCTGGGTACTTTTATCTGTCCCCTCCACCCCA。

[0354] SEQ ID NO:20 - AAVS1 Intron 1 Targeting Vector - Left Homology Arm (CHr 19: 55115776-55116775 Human GRCh38 - Ensembl Version 104 - May 2021)

[0355]

[0356] SEQ ID NO:21 - AAVS1 Intron 1 Targeting Vector - Right Homology Arm (Chr 19: 55114775-55115775 Human GRCh38 - Ensembl Version 104 - May 2021)

[0357]

[0358] SEQ ID NO:22 MAGE-B2 / A4 ADB959 knock-in into PTPRC exon 33 TCR landing pad - left homology arm Chr1: 198,754,605-198,756,226 (human GRCh38 - Ensembl version 104 - May 2021)

[0359]

[0360] SEQ ID NO:23 MAGE-B2 / A4 ADB959 knock-in into PTPRC exon 33 TCR landing pad - right homology arm; includes TRAC domain sequence (nucleotides 1-396), BGH polyA signal (397-621) and nucleotides corresponding to chromosome 1: 198,756,132-198,757,230 (human GRCh38 - Ensembl version 104 - May 2021)

[0361] AGTTCAGGTTCAAGAGCTAAAAGGAGCGGATCAGGT

[0362] SEQ ID NO:24 - Furin SG linker

[0363] GGCAGCCGGGCCAAGAGATCTGGATCCGGC

[0364] SEQ ID NO:25—Furin SG linker

[0365] SSGSRAKRSGS

[0366] SEQ ID NO:26 Furin SG linker

[0367] GSRAKRSGSG

[0368] SEQ ID NO:27 Furin SG linker

[0369] GAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAAAACCCTGGCCCT

[0370] SEQ ID NO:28—T2A skipping-like sequence nucleotide sequence

[0371] EGRGSLLTCGDVEENPGP

[0372] SEQ ID NO:29—T2A skipping-like sequence amino acid sequence

[0373] GCTACCAACTTTAGCCTGCTGAAGCAGGCCGGGGACGTGGAAGAAAACCCTGGCCCT

[0374] SEQ ID NO:30—P2A jumping-like sequence nucleotide sequence

[0375] ATNFSLLKQAGDVEENPGP

[0376] Amino acid sequence of the SEQ ID NO:31 P2A-like sequence

[0377] ATGGCCAGCCTGCTGTTCTTCTGCGGCGCCTTCTACCTGCTGGGCACCGGCTCTATGGATGCCGACGTGACCCAGACCCCCCGGAACAGAATCACCAAGACCGGCAAGCGGATCATGCTGGAATGCTCCCAGACCAAGGGCCACGACCGGATGTACTGGTACAGACAGGACCCTGGCCTGGGCCTGCGGCTGATCTACTACAGCTTCGACGTGAAGGACATCAACAAGGGCGAGATCAGCGACGGCTACAGCGTGTCCAGACAGGCTCAGGCCAAGTTCAGCCTGTCCCTGGAAAGCGCCATCCCCAACCAGACCGCCCTGTACTTTTGTGCCACAAGCGGCCAGGGCGCCTACAACGAGCAGTTCTTTGGCCCTGGCACCCGGCTGACAGTGCTGGAAGATCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAAATCAGCCACACCCAGAAAGCCACACTCGTGTGTCTGGCCACCGGCTTCTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTGTCCACCGATCCCCAGCCTCTGAAAGAACAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGTGCAGTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCCCGTGACACAGATCGTGTCTGCCGAAGCTTGGGGGCGCGCCGATTGTGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGAAAGGCCACACTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC

[0378] SEQ ID NO: 32 - Nucleotide sequence of the TCRβ chain of ADB959

[0379] ATGAAGAAGCACCTGACCACCTTTCTCGTGATCCTGTGGCTGTACTTCTACCGGGGCAACGGCAAGAACCAGGTGGAACAGAGCCCCCAGAGCCTGATCATCCTGGAAGGCAAGAACTGCACCCTGCAGTGCAACTACACCGTGTCCCCCTTCAGCAACCTGCGGTGGTACAAGCAGGACACCGGCAGAGGCCCTGTGTCCCTGACCATCGTGACCTTCAGCGAGAACACCAAGAGCAACGGCCGGTACACCGCCACCCTGGACGCCGATACAAAGCAGAGCAGCCTGCACATCACCGCCAGCCAGCTGAGCGATAGCGCCAGCTACATCTGCGTGGTGTCCGGCGGCACAGACAGCTGGGGCAAGCTGCAGTTTGGCGCCGGAACACAGGTGGTCGTGACCCCCGACATCCAGAACCCTGACCCTGCAGTATATCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA

[0380] SEQ ID NO: 33 - Nucleotide sequence of the TCRα chain of ADB959

[0381] EQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNGPASPALNQGSSSGSRAKRSGSGEGRGSLLTCGDVEENPGPMASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSRAKRSGSGATNFSLLKQAGDVEENPGPMKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIVTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0382] SEQ ID NO:34 - Translated sequence of PTPRC exon

[0383] 33_T2A_ADB959_TCRβ_P2A_TCRα

[0384]

[0385] SEQ ID NO:35 - MAGE-B2 / A4 ADB959 knock-in into AAVS1 TCR landing pad - left homology arm (Chr19: 55,115,701-55,117,349 human GRCh38 - Ensembl version 104 - May 2021)

[0386] GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG

[0387] SEQ ID NO:36—EF1A short promoter

[0388] ATGGCCAGCCTGCTGTTCTTCTGCGGCGCCTTCTACCTGCTGGGCACCGGCTCTATGGATGCCGACGTGACCCAGACCCCCCGGAACAGAATCACCAAGACCGGCAAGCGGATCATGCTGGAATGCTCCCAGACCAAGGGCCACGACCGGATGTACTGGTACAGACAGGACCCTGGCCTGGGCCTGCGGCTGATCTACTACAGCTTCGACGTGAAGGACATCAACAAGGGCGAGATCAGCGACGGCTACAGCGTGTCCAGACAGGCTCAGGCCAAGTTCAGCCTGTCCCTGGAAAGCGCCATCCCCAACCAGACCGCCCTGTACTTTTGTGCCACAAGCGGCCAGGGCGCCTACAACGAGCAGTTCTTTGGCCCTGGCACCCGGCTGACAGTGCTGGAAGATCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAAATCAGCCACACCCAGAAAGCCACACTCGTGTGTCTGGCCACCGGCTTCTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTGTCCACCGATCCCCAGCCTCTGAAAGAACAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGTGCAGTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCCCGTGACACAGATCGTGTCTGCCGAAGCTTGGGGGCGCGCCGATTGTGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGAAAGGCCACACTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC

[0389] SEQ ID NO:37 - Nucleotide sequence of ADB959 TCRβ chain

[0390] ATGAAGAAGCACCTGACCACCTTTCTCGTGATCCTGTGGCTGTACTTCTACCGGGGCAACGGCAAGAACCAGGTGGAACAGAGCCCCCAGAGCCTGATCATCCTGGAAGGCAAGAACTGCACCCTGCAGTGCAACTACACCGTGTCCCCCTTCAGCAACCTGCGGTGGTACAAGCAGGACACCGGCAGAGGCCCTGTGTCCCTGACCATCGTGACCTTCAGCGAGAACACCAAGAGCAACGGCCGGTACACCGCCACCCTGGACGCCGATACAAAGCAGAGCAGCCTGCACATCACCGCCAGCCAGCTGAGCGATAGCGCCAGCTACATCTGCGTGGTGTCCGGCGGCACAGACAGCTGGGGCAAGCTGCAGTTTGGCGCCGGAACACAGGTGGTCGTGACCCCCGACATCCAGAACCCTGACCCTGCAGTATATCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA

[0391] SEQ ID NO:38 - Nucleotide sequence of ADB959 TCR α - chain

[0392] MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSRAKRSGSGATNFSLLKQAGDVEENPGPMKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIVTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS-

[0393] SEQ ID NO:39 - ADB959 TCRΒ_P2A_TCRΑ Amino Acid Sequence

[0394] CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

[0395] SEQ ID NO:40 - BGH POLY A Signal

[0396]

[0397] SEQ ID NO:41 - MAGE-B2 / A4 ADB959 knock-in into AAVS1 TCR landing pad - right homology arm - contains TRAC domain sequence (nucleotides 1-396), BGH polyA signal (397-621) and nucleotides corresponding to Chr19: 55,114,725-55,115,825 (human GRCh38 - Ensembl version 104 - May 2021)

[0398] 5'- TTCAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCA GGCTCCGGTGCCCGTCA-3'

[0399] SEQ ID NO:42 (underlined B2M FWD target sequence)

[0400] 5'- TGACTTTCCATTCTCTGCTGGATGACGTGAGTAAACCTGAA AACTTGTTTATTGCAGCTTATAATGG-3'

[0401] SEQ ID NO:43 (underlined B2M FWD target sequence)

[0402] 5'CGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCTGGGTTCCCTTTTCCTTC 3'

[0403] SEQ ID NO:44 (FWD; binds to Chr19: 55116254-55116274GRCh38.p14)

[0404] 5'-GACTTTCCATTCTCTGCTGGATGACGTGAGTAAACCTGAATGTGGGGTGGAGGGGACAG–3'

[0405] SEQ ID NO:45 (REV binds Chr 19:55115774-55115792GRCh38.p14).

[0406] 5'-TCAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAGTGGGGCCACTAGGGACAGGATTG-3'SEQ ID NO: 46 (FWD binds Chr19: 55115750-55115773GRCh38.p14)

[0407] 5'-GAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCTACTGGCCTTATCTCACAG-3'

[0408] SEQ ID NO:47 (REV binds to Chr19: 55115274 - 55115292 GRCh38.p14)

[0409] 5'GTCGATCCTACCATCCACTCGACACACCCGCCAGCGGCCGCTGCCAAGCTTGCCGCCACCATGAAGAAGCACCTGACCACCTTTCTCGTGATC-3'

[0410] SEQ ID NO:48 (FWD)

[0411] 5'-CCAATCCTGTCCCTAGTGGCCCCACTGACTTTCCATTCCCATAGAGCCCACCGCATCCCCAG-3'SEQ ID NO:49 (REV)

[0412] 5'CGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCTGGGTTCCCTTTTCCTTC-3'

[0413] SEQ ID NO:50 (FWD)

[0414] 5'GTGGGCGATGTGCGCTCTGCCCACTGACGGGCACCGGAGCCTCTGCTGGATGACGTGAGTAAACCTGAATGTGGGGTGGAGGGGACAG–3'

[0415] SEQ ID NO:51 (REV)

[0416] 5'-GAATGGAAAGTCAGTGGGGCCACTAGGGACAGGATTGG-3'

[0417] SEQ ID NO:52 (FWD)

[0418] 5'TTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCTACTGGCCTTATCTCACAG-3'

[0419] SEQ ID NO:53 (REV)

[0420] 5'-GGATGCTCTTTCCGGAGCAC-3'

[0421] SEQ ID NO:54 (5'FWD; binds Chr19: 55116402-55116383GRCh38.p14) 5'-GCACCGGTTCAATTGCCGAC-3'

[0422] SEQ ID NO:55 (5'REV; EF1-α binding to ADB00794_001)

[0423] 5'-TGGTGAACACCTAGGACGCA-3'

[0424] SEQ ID NO:56 (3'FWD; binds to Chr19: 55115182-55115201GRCh38.p14)

[0425] 5'-GGCTCTCGGAGAATGACGA-3'

[0426] SEQ ID NO:57 (3'REV; A2M10 binding to ADB00794_001)

[0427]

[0428]

[0429] SEQ ID NO: 58 (A2M10 TCR cassette ADB00794_001 Underline = EF-1α promoter; Dotted underline = SV40 polyadenylation signal Dashed underline = A2M10 TCR Double underline = Furin cleavage site, P2A)

[0430]

[0431]

[0432]

[0433]

[0434] SEQ ID NO:59 (plasmid containing A2M10 placeholder TCR cassette (ADB00794_001) Full underline = homology arms; wavy underline = B2M target site; dotted underline = A2M10 TCR dotted underline = EF-1α promoter; double underline = furin cleavage site, P2A; dotted underline = SV40 polyA signal)

[0435] MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVRGTGRRALTFGSGTRLQVQPNIQNPDPAVYQLRDSKSSDK SVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSGSRAKRSGSGATNFSLLKQAGDVEENPGPRMS LGLLCCGVFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPMGLRLIHYSVAEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSFTDTQYFPGGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQK ATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0436] SEQ ID NO:60 (Protein sequence of A2M10 placeholder TCR)

[0437]

[0438]

[0439]

[0440]

[0441] SEQ ID NO:61 (plasmid containing the A2M4 exchange TCR cassette (ADB01032_026) Dashed underline = homology arms; full underline = EF-1α promoter; dotted underline = A2M10 TCR; double underline = furin cleavage site, P2A; dotted underline = BGH polyA signal)

[0442] MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFGAGTQVVVTPDIQNPDPAVYQLRDSKSSD KSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSGSRAKRSGSGATNFSLLKQAGDVEENPGPRMA SLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYEEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHT QKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0443] SEQ ID NO:62 (protein sequence of A2M4 exchange TCR)

[0444] UCACGUCAUCCAGCAGAGAA

[0445] SEQ ID NO:63 (CRISPR-Cas9 guide RNA)

[0446] CTGGGTTCCCTTTTCCTTCTCCTTCTGGGGCCTGTGCCATCTCTCGTTTCTTAGGATGGCCTTCTCCGACGGATGTCTCCCTTGCGTCCCGCCTCCCCTTCTTGTAGGCCTGCATCATCACCGTTTTTCTGGACAACCCCAAAGTACCCCGTCTCCCTGGCTTTAGCCACCTCTCCATCCTCTTGCTTTCTTTGCCTGGACACCCCGTTCTCCTGTGGATTCGGGTCACCTCTCACTCCTTTCATTTGGGCAGCTCCCCTACCCCCCTTACCTCTCTAGTCTGTGCTAGCTCTTCCAGCCCCCTGTCATGGCATCTTCCAGGGGTCCGAGAGCTCAGCTAGTCTTCTTCCTCCAACCCGGGCCCCTATGTCCACTTCAGGACAGCATGTTTGCTGCCTCCAGGGATCCTGTGTCCCCGAGCTGGGACCACCTTATATTCCCAGGGCCGGTTAATGTGGCTCTGGTTCTGGGTACTTTTATCTGTCCCCTCCACCCCACA

[0447] SEQ ID NO:64 - Plasmid ADB00794_001 Left Homologous Arm (Chr 19: 55115774 to 55116272 Human GRCh38.p14 Primary Assembly

[0448]

[0449] SEQ ID NO: 65 - EF1α promoter DNA sequence

[0450] ATGATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTCCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCGTGAGAGGCACGGGCAGGAGAGCACTTACTTTTGGGAGTGGAACAAGACTCCAAGTGCAACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC

[0451] SEQ ID NO: 66 - A2M10 c794 TCRα - chain nucleotide sequence (from ADB00794_001)

[0452] MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVRGTGRRALTFGSGTRLQVQPNIQN PDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0453] SEQ ID NO:67 - A2M10 c794 alpha chain amino acid sequence (from ADB00794_001)

[0454] GGCAGCCGGGCCAAGAGAAGCGGATCCGGC

[0455] SEQ ID NO:68—Furin SG linker nucleotide sequence

[0456] GCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAAAACCCTGGCCCTAGG

[0457] SEQ ID NO:69—P2A jumping-like sequence nucleotide sequence

[0458] ATNFSLLKQAGDVEENPGPR

[0459] SEQ ID NO:70—P2A jumping-like sequence amino acid sequence

[0460] ATGAGCCTCGGGCTCCTGTGCTGTGGGGTGTTTTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCGGGTCCTGAAGACAGGACAGAGCATGACACTGCTGTGTGCCCAGGATATGAACCATGAATACATGTACTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGCCGAGGGTACAACTGCCAAAGGAGAGGTCCCTGATGGCTACAATGTCTCCAGATTAAAAAAACAGAATTTCCTGCTGGGGTTGGAGTCGGCTGCTCCCTCCCAAACATCTGTGTACTTCTGTGCCAGCAGTTTCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC

[0461] SEQ ID NO: 71 - A2M10 c794 TCRβ chain nucleotide sequence (from ADB00794_001)

[0462] MSLGLLCCGVFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVAEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSFTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0463] SEQ ID NO:72 - A2M10 c794 TCRβ amino acid sequence (from ADB00794_001)

[0464] TAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTT

[0465] SEQ ID NO:73 - SV40 polyA signal nucleotide sequence

[0466] GTGGGGCCACTAGGGACAGGATTGGTGACAGAAAAGCCCCATCCTTAGGCCTCCTCCTTCCTAGTCTCCTGATATTGGGTCTAACCCCCACCTCCTGTTAGGCAGATTCCTTATCTGGTGACACA CCCCCATTTCCTGGAGCCATCTCTCTCCTTGCCAGAACCTCTAAGGTTTGCTTACGATGGAGCCAGAGGATCCTGGGAGGGAGAGCTTGGCAGGGGGTGGGAGGGAAGGGGGGGATGCGTGAC CTGCCCGGTTCTCAGTGGCCACCCTGCGCTACCCTCTCCCAGAACCTGAGCTGCTCTGACGCGGCTGTCTGGTGCGTTTCACTGATCCTGGTGCTGCAGCTTCCTTACACTTCCCAAGAGGAGAA GCAGTTTGGAAAAACAAAATCAGAATAAGTTGGTCCTGAGTTCTAACTTTGGCTCTTCACCTTTCTAGTCCCCAATTTATATTGTTCCTCCGTGCGTCAGTTTTACCTGTGAGATAAGGCCAGTA

[0467] SEQ ID NO:74—Plasmid ADB00794_001 right homology arm (Chr 19: 55115274 to 55115773 human GRCh38.p14 primary assembly)

[0468] GACTTTCCA TTCTCTGCTGGATGACGTGA GTAAACCTGAATGTGGGGTGGAGGGGACAG

[0469] SEQ ID NO:75—Reverse primer used to amplify the left homology arm of Strategy 1 plasmid ADB00794_001 (binds Chr19: 55115774-55115792GRCh38.p14). Underline—B2M sgRNA target site.

[0470]

[0471]

[0472]

[0473] SEQ ID NO:76 - Nucleotide sequence of A2M10 TCR cassette. Underline = EF-1α promoter. Dotted underline = SV40 polyadenylation signal. Dashed underline = A2M10 TCR. Double underline = furin cleavage site, P2A

[0474] GCTGGGTTCCCTTTTTCCTTCTCCTTCTGGGGCCTGTGCCATCTCTCGTTTCTTAGGATGGCCTTCTCCGACGGATGTCTCCCTTGCGTCCCGCCTCCCCTTCTTGTAGGCCTGCATCACCGTTTTTCTG GACAACCCCAAAGTACCCCGTCTCCCTGGCTTTAGCCACCTCTCCATCCTCTTGCTTTCTTTGCCTGGACACCCCGTTCTCCTGTGGATTCGGGTCACCTCTCACTCCTTTCATTTGGGCAGCTCCCCTACC CCCCTTACCTCTCTAGTCTGTGCTAGCTCTCCAGCCCCCTGTCATGGCATCTTCCAGGGGTCCGAGAGCTCAGCTAGTCTTCTTCCTCCAACCCGGGCCCCTATGTCCACTTCAGGACAGCATGTTTGCTG CCTCCAGGGATCCTGTGTCCCCGAGCTGGGACCACCTTATATTCCCAGGGCCGGTTAATGTGGCTCTGGTTCTGGGTACTTTTATCTGTCCCCTCCACCCCACATTCAGGTTTACTCACGTCATCCAGCAGA

[0475] SEQ ID NO:77—Plasmid ADB01032_026 left homology arm (human Chr19: 55115774 to 55116273, Chr15: 44715434 to 44715462GRCh38.p14 primary assembly)

[0476] ATGAAGAAGCACCTGACCACCTTTCTCGTGATCCTGTGGCTGTACTTCTACCGGGGCAACGGCAAGAACCAGGTGGAACAGAGCCCCCAGAGCCTGATCATCCTGGAAGGCAAGAACTGCACCCTGCAGTGCAACTACACCGTGTCCCCCTTCAGCAACCTGCGGTGGTACAAGCAGGACACCGGCAGAGGCCCTGTGTCCCTGACCATCCTGACCTTCAGCGAGAACACCAAGAGCAACGGCCGGTACACCGCCACCCTGGACGCCGATACAAAGCAGAGCAGCCTGCACATCACCGCCAGCCAGCTGAGCGATAGCGCCAGCTACATCTGCGTGGTGTCCGGCGGCACAGACAGCTGGGGCAAGCTGCAGTTTGGCGCCGGAACACAGGTGGTCGTGACCCCCGACATCCAGAACCCTGACCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAATAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTCAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGACTGTGG

[0477] SEQ ID NO:78 - A2M4 c1032 TCRα chain nucleotide sequence (from ADB01032_026)

[0478] MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFGAGTQVVVTPDI QNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLW

[0479] SEQ ID NO:79-A2M4 c1032 TCR alpha chain amino acid sequence (from ADB01032_026)

[0480] GCTACCAACTTTAGCCTGCTGAAGCAGGCCGGGGACGTGGAAGAAAACCCTGGCCCTAGG

[0481] SEQ ID NO:80—P2A jumping-like sequence nucleotide sequence

[0482] ATGGCCAGCCTGCTGTTCTTCTGCGGCGCCTTCTACCTGCTGGGCACCGGCTCTATGGATGCCGACGTGACCCAGACCCCCCGGAACAGAATCACCAAGACCGGCAAGCGGATCATGCTGGAATGCTCCCAGACCAAGGGCCACGACCGGATGTACTGGTACAGACAGGACCCTGGCCTGGGCCTGCGGCTGATCTACTACAGCTTCGACGTGAAGGACATCAACAAGGGCGAGATCAGCGACGGCTACAGCGTGTCCAGACAGGCTCAGGCCAAGTTCAGCCTGTCCCTGGAAAGCGCCATCCCCAACCAGACCGCCCTGTACTTTTGTGCCACAAGCGGCCAGGGCGCCTACGAGGAGCAGTTCTTTGGCCCTGGCACCCGGCTGACAGTGCTGGAAGATCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAAATCAGCCACACCCAGAAAGCCACACTCGTGTGTCTGGCCACCGGCTTCTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTGTCCACCGATCCCCAGCCTCTGAAAGAACAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGTGCAGTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCCCGTGACACAGATCGTGTCTGCCGAAGCTTGGGGGCGCGCCGATTGTGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGAAAGGCCACACTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGCTAA

[0483] SEQ ID NO:81 - A2M4 c1032 TCRβ chain nucleotide sequence (from ADB01032_026)

[0484] MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYEEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0485] SEQ ID NO:82 - A2M4 c1032 TCRβ amino acid sequence (from ADB01032_026)

[0486] GAATGGAAAGTCAGTGGGGCCACTAGGGACAGGATTGGTGACAGAAAAGCCCCATCCTTAGGCCTCCTCCTTCCTAGTCTCCTGATATTGGGTCTAACCCCCACCTCCTGTTAGGCAGATTCCTTATCTGGTGACACACCCCCATTTCCTGGAGCCATCTCTCTCCTTGCCAGAACCTCTAAGGTTTGCTTACGATGGAGCCAGAGAGGATCCTGGGAGGGAGAGCTTGGCAGGGGGTGGGAGGGAAGGGGGGGATGCGTGACCTGCCCGGTTCTCAGTGGCCACCCTGCGCTACCCTCTCCCAGAACCTGAGCTGCTCTGACGCGGCTGTCTGGTGCGTTTCACTGATCCTGGTGCTGCAGCTTCCTTACACTTCCCAAGAGGAGAAGCAGTTTGGAAAAACAAAATCAGAATAAGTTGGTCCTGAGTTCTAACTTTGGCTCTTCACCTTTCTAGTCCCCAATTTATATTGTTCCTCCGTGCGTCAGTTTTACCTGTGAGATAAGGCCAGTA

[0487] SEQ ID NO:83-Plasmid ADB01032_026 right homology arm (human Chr19: 55115274 to

[0488] 55115775GRCh38.p14 primary assembly)

[0489] GGCTCCGGTGCCCGTCAGTGGGC

[0490] SEQ ID NO:84—Forward primer for amplification of EF1α promoter

[0491] GGTGGCGGCAAGCTTGGCAGCGGC

[0492] SEQ ID NO:85—Reverse primer for amplifying the EF1α promoter

Claims

1. A method for producing immune cells expressing a therapeutic antigen receptor, the method comprising: (i) providing an immune cell comprising a heterologous expression cassette, Wherein the expression cassette comprises: (a) a coding sequence for producing a T cell receptor (TCR), (b) a constitutive promoter operably linked to the coding sequence, (c) target site, and (ii) replacing said heterologous expression cassette in said immune cell with an expression construct comprising a coding sequence for a therapeutic antigen receptor, The therapeutic antigen receptor is allowed to be expressed in the immune cell.

2. The method of claim 1, wherein the therapeutic antigen receptor specifically binds to MHC displaying a peptide fragment of a target antigen expressed by a cell, or specifically binds to a target antigen or a peptide thereof expressed by a cell independently of MHC presentation.

3. The method of claim 2, wherein the therapeutic antigen receptor specifically binds to MHC displaying a peptide fragment of a tumor antigen expressed by cancer cells, or specifically binds to a tumor antigen expressed by cancer cells or a peptide fragment thereof independent of MHC presentation.

4. The method according to any one of the preceding claims, wherein the immune cell is a T cell.

5. The method according to any one of the preceding claims, wherein the heterologous expression cassette in the immune cell is replaced by a method comprising: introducing a nucleic acid molecule comprising the expression construct and a 5' homology arm into the immune cell, wherein the 5' homology arm is complementary to the 5' targeting site of the heterologous expression cassette, The expression construct is allowed to replace the expression cassette in the genome of the immune cell.

6. The method of claim 5, wherein the method further comprises introducing a vector comprising a nucleic acid into the immune cell, wherein the nucleic acid encodes CRISPR / Cas9 targeting the targeting site.

7. The method according to any one of the preceding claims, wherein the targeting site is a 5' targeting site.

8. The method of claim 7, wherein the cassette further comprises a 3' targeting site.

9. The method of claim 8, wherein the heterologous expression cassette in the immune cell is replaced by a method comprising: introducing into the immune cell a nucleic acid molecule, such as a DNA molecule, comprising an expression construct flanked by 5' and 3' homology arms, wherein the 5' and 3' homology arms are complementary to the 5' and 3' targeting sites of the heterologous expression cassette, The expression construct is allowed to replace the expression cassette in the genome of the immune cell.

10. The method of claim 9, wherein the method further comprises introducing a vector comprising a nucleic acid into the immune cell, the nucleic acid encoding CRISPR / Cas9 targeting the 5' and 3' targeting sites.

11. The method of any one of the preceding claims, wherein the therapeutic antigen receptor is a T cell receptor (TCR).

12. The method of claim 11, wherein the therapeutic TCR specifically binds to cancer cells.

13. The method according to any one of the preceding claims, wherein the expression cassette comprises 5' and 3' targeting sites, and the targeting sites comprise nucleotide sequences from the constant region of the TCR alpha chain.

14. The method of claim 13, wherein the 3' targeting site is located within the coding sequence for the generation of the TCR.

15. The method according to any one of the preceding claims, wherein the immune cell comprising the heterologous expression cassette is produced by a method comprising: (i) transfecting an iPSC with a nucleic acid comprising a heterologous expression cassette such that the cassette is integrated into the genome of the iPSC, wherein the expression cassette comprises (a) a coding sequence for producing a T cell receptor (TCR), (b) a constitutive promoter operably linked to the coding sequence, and (c) target site, and (ii) differentiating the iPSCs into immune cells comprising the heterologous expression cassette.

16. The method of claim 15, wherein the targeting site is a 5' targeting site.

17. The method of claim 16, wherein the cassette further comprises a 3' targeting site.

18. The method of claim 15, 16 or 17, wherein the iPSCs are differentiated into immune cells by a method comprising: (i) differentiating the iPSCs into mesoderm cells, (ii) differentiating the mesodermal cells into hemogenic endothelial cells, (iii) differentiating the hemogenic endothelial cells into a HPC population, (iv) differentiating the HPCs into immune cell progenitor cells; and (v) maturing the progenitor immune cell population to generate an immune cell population.

19. The method according to claim 18, wherein the iPSCs are cultured in a first mesoderm induction medium, a second mesoderm induction medium, and a third mesoderm induction medium in sequence to induce differentiation into mesoderm cells, The first mesoderm induction medium consists of a chemically defined nutrient medium supplemented with one or more differentiation factors, wherein the one or more differentiation factors consist of activin, The second mesoderm induction medium consists of a chemically defined nutrient medium supplemented with one or more differentiation factors, wherein the one or more differentiation factors consist of activin, BMP and FGF, and The third mesoderm induction medium consists of a chemically defined nutrient medium supplemented with one or more differentiation factors, wherein the one or more differentiation factors consist of activin, BMP, FGF and a GSK3 inhibitor.

20. The method according to claim 18 or 19, wherein the mesodermal cells are cultured in a HE induction medium to induce differentiation into HECs; the HE induction medium consists of a chemically defined nutrient medium supplemented with one or more differentiation factors, wherein the one or more differentiation factors consist of SCF and VEGF.

21. The method according to any one of claims 18 to 20, wherein the HECs are cultured in a hematopoietic induction medium to induce differentiation into HPCs; the hematopoietic induction medium consists of a chemically defined nutrient medium supplemented with one or more differentiation factors, wherein the one or more differentiation factors consist of: VEGF, SCF, thrombopoietin (TPO), Flt3 ligand (Flt3L), IL-3, IL-6, IL-7, IL-11, IGF-1, BMP, FGF, sonic hedgehog (SHH), erythropoietin (EPO), angiotensin II and angiotensin II type 1 receptor (AT1) antagonist.

22. The method of any one of claims 18 to 21, wherein the HPCs are cultured in a lymphoid expansion medium to produce the progenitor immune cells; the lymphoid expansion medium consisting of a chemically defined nutrient medium supplemented with one or more differentiation factors, wherein the one or more differentiation factors consist of SCF, FLT3L, TPO and IL7.

23. The method of any one of claims 18 to 22, wherein the progenitor immune cells are matured by a method comprising: culturing the progenitor immune cell colony in a maturation medium to produce the immune cells; the maturation medium consisting of a chemically defined nutrient medium supplemented with one or more differentiation factors, wherein the one or more differentiation factors consist of SCF, FLT3L and IL7.

24. The method of any one of the preceding claims, comprising concentrating the population of immune cells expressing the therapeutic antigen receptor.

25. A method according to any one of the preceding claims, comprising storing the population of immune cells expressing the therapeutic antigen receptor.

26. The method of any one of the preceding claims, comprising formulating the population of immune cells expressing the therapeutic antigen receptor with a pharmaceutically acceptable excipient.

27. A population of immune cells expressing one or more therapeutic antigen receptors produced by the method of any one of claims 1 to 26.

28. A pharmaceutical composition comprising a population of immune cells expressing one or more therapeutic antigen receptors produced by the method of any one of claims 1 to 26 and a pharmaceutically acceptable excipient.

29. An immune cell population expressing one or more therapeutic antigen receptors produced by the method of any one of claims 1 to 26, for use in a therapeutic method.

30. A population of immune cells expressing one or more therapeutic antigen receptors produced by the method of any one of claims 1 to 26, for use in a method of treating cancer.

31. A method of treating cancer, the method comprising administering to an individual in need thereof a population of immune cells expressing one or more therapeutic antigen receptors produced by the method of any one of claims 1 to 26.

32. Use of an immune cell population expressing one or more therapeutic antigen receptors produced by the method according to any one of claims 1 to 26 for the preparation of a medicament for treating cancer.

33. An immune cell comprising a heterologous expression cassette integrated into its genome, wherein the expression cassette comprises (a) a coding sequence for producing a T cell receptor (TCR), (b) a constitutive promoter operably linked to the coding sequence, and (c) Targeting site.

34. The immune cell of claim 31, wherein the targeting site is a 5' targeting site.

35. The immune cell of claim 34, wherein the cassette further comprises a 3' targeting site.

36. An iPSC comprising a heterologous expression cassette integrated into its genome, wherein the expression cassette comprises (a) a coding sequence for producing a T cell receptor (TCR), (b) a constitutive promoter operably linked to the coding sequence, and (c) Targeting site.

37. The iPSC of claim 33, wherein the targeting site is a 5' targeting site.

38. The iPSC of claim 37, wherein the cassette further comprises a 3' targeting site.

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