CD70 CAR-T compositions and methods for cell-based therapy

Abstract

The compositions and methods described herein relate to the use of a CRISPR / Cas9 system in combination with a CAR technology for improving the activity of anti-CD70 CAR-T cells. The engineered cells may comprise a genetic modification in one or more of the HLA-A, HLA-B, TRAC, CIITA, TGFBR2, or CD70 genes.

Description

Cross-reference of related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 519,551, filed August 14, 2023; U.S. Provisional Application No. 63 / 610,582, filed December 15, 2023; and U.S. Provisional Application No. 63 / 659,675, filed June 13, 2024, pursuant to 35 USC 119(e), the contents of each of which are incorporated herein by reference in their entirety.

[0002] Reference to electronic sequence listing This application contains a sequence list, which has been electronically submitted in XML file format and is hereby incorporated in its entirety by reference. The XML file was created on August 12, 2024, named "01155-0059-00PCT.xml", and has a size of 3,148,975 bytes. Summary of the Invention

[0003] This disclosure generally describes chimeric antigen receptor (CAR) reagents (e.g., cells and compositions) and methods. This disclosure relates to the use of CAR technology in conjunction with a CRISPR system for delivering CAR-transduced immune effector cells (e.g., T cells, NK cells).

[0004] CD70 is a transmembrane protein that is transiently expressed on the surface of CD4+ and CD8+ T cells, regulatory T cells (Tregs), B cells, antigen-presenting cells (such as dendritic cells), and natural killer (NK) cells in response to immune activation.

[0005] Although CD70 expression is tightly controlled in normal tissues, many cancer cell types exhibit high levels of CD70. For example, CD70 is overexpressed in both solid and liquid cancer types, such as renal cell carcinoma (RCC), acute myeloid leukemia (AML), Hodgkin's lymphoma and non-Hodgkin's lymphoma, multiple myeloma, pancreatic cancer, ovarian cancer, and non-small cell lung cancer (NSCLC). Notably, many of these cancer types have proven difficult to treat, resulting in a significant unmet need for novel treatment methods. Due to its differential expression on the surface of healthy and cancer cells, CD70 represents an attractive target for emerging therapies, including immunotherapy.

[0006] Despite its promising therapeutic potential, anti-CD70 CAR-T therapy faces numerous logical and technical hurdles. Current CAR-T protocols are based on autologous cell transfer. In this approach, T cells recovered from a patient are genetically modified in vitro and cultured before being infused into the patient. This method, utilizing the patient's own lymphocytes, reduces the risk of rejection. However, so-called autologous therapy relies on the availability of functional lymphocytes, which can be compromised by previous treatment lines. Furthermore, each patient's autologous cell preparation is essentially a new product, leading to substantial variations in safety and efficacy.

[0007] "Off-the-shelf" therapies using donor (allogeneic) cells eliminate the need for restoring and modifying the patient's own lymphocytes. However, allogeneic CAR-T cells can elicit unwanted immune responses or otherwise persist transiently. Typically, immune rejection of allogeneic cells is caused by a mismatch of major histocompatibility complex (MHC) molecules between the donor and recipient. For example, slight differences in MHC alleles between individuals can lead to T cell activation in the recipient. During T cell development, an individual's T cell lineage is tolerant to its own MHC molecules, but T cells that recognize the MHC molecules of another individual can persist in circulation and are called allogeneic reactive T cells. Allogeneic reactive T cells can be activated, for example, by the presence of cells from another individual expressing MHC molecules in the body, leading to conditions such as graft-versus-host disease and transplant rejection.

[0008] Methods and compositions for reducing the susceptibility of allogeneic anti-CD70 CAR-T cells to rejection or for improving the activity of anti-CD70 CAR-T cells are of interest.

[0009] Therefore, there is a need for improved methods and compositions for modifying anti-CD70 CAR-T cells to overcome recipient immune rejection and improve the activity of anti-CD70 CAR-T cells. This disclosure provides genome editing of anti-CD70 CAR-T cells using a CRISPR / Cas9 system.

[0010] The engineered cells contain gene modifications in the HLA-A, HLA-B, TRAC, CIITA (trans-activator of major histocompatibility complex class II), TGFBR2, or CD70 genes, and can be used for cell therapy. This disclosure further provides compositions and methods for reducing or eliminating the surface expression of endogenous T cell receptors, MHC class I or II proteins, CD70, or TGFBR2 in cells by gene modification of the HLA-A, HLA-B, TRAC, CIITA, TGFBR2, or CD70 loci.

[0011] In some embodiments, a method is provided for reducing the surface expression of HLA-A protein in engineered cells relative to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments provided herein. In some embodiments, a method is provided for reducing the surface expression of HLA-B protein in engineered cells relative to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments provided herein. In some embodiments, a method is provided for reducing the surface expression of TRAC protein in engineered cells relative to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments provided herein. In some embodiments, a method is provided for reducing the surface expression of MHC class II proteins in engineered cells relative to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments provided herein. In some embodiments, a method is provided for reducing the surface expression of TGFBR2 protein in engineered cells relative to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments provided herein. In some embodiments, a method is provided for reducing the surface expression of CD70 protein in engineered cells relative to unmodified cells, the method comprising contacting the cells with a composition of any of the embodiments provided herein. Attached Figure Description

[0012] Figure 1 A schematic diagram of an anti-CD70 CAR construct with an alternative antigen-binding protein and a co-stimulatory domain is shown.

[0013] Figure 2A-2D Four anti-CD70 CAR constructs were demonstrated to re-attack the 786-O tumor cell line in vitro, based on the area of ​​cancer cells (mm²). 2 This is used to measure and compare with a benchmark construct. Figure 2A The re-attack results of constructs 5719 and 5281 are shown. These constructs share the same scFv antigen-binding protein domain, but some intracellular domains are different. Figure 2B The re-attack results of constructs 5284 and 5718 are shown. These constructs share the same scFv antigen-binding protein domain, but some intracellular domains are different. Figure 2C The re-attack results of constructs 5283 and 5717 are shown. These constructs share the same scFv antigen-binding protein domain, but some intracellular domains are different. Figure 2D The results of re-attacks on construct 5715, which has a 41BB co-stimulatory domain, are shown.

[0014] Figure 3The efficacy of five anti-CD70 CAR constructs in the 786-O mouse tumor cell model was compared with that of the baseline construct, based on tumor volume (mm). 3 The measurements were taken at a dose of 3e6. Constructs 5715, 5717, 5719, and 5718, each containing a 41BB co-stimulatory domain, and construct 5281, containing a CD28 co-stimulatory domain, were tested.

[0015] Figure 4 The efficacy of two anti-CD70 CAR constructs versus the baseline construct in a 786-O mouse tumor cell model was compared, based on tumor volume (mm²). 3 The measurements were taken using a 1e6 dose. Constructs 5719 and 5715 were tested.

[0016] Figures 5A-5D The effects of dual (DKO) versus single (SKO) immune enhancement editing (IEE) knockout on the 786-O tumor cell line using constructs 5719, 5718, or 4645 are shown, measured as the percentage of remaining viable tumor cells. Unedited cells served as a control. Constructs 5719 and 5718 were tested individually, with CD70 SKO, TGFβR2 SKO, and CD70+ TGFβR2 DKO. Figure 5A The percentage of tumor cell viability in construct 5719 in the absence of TGFβ is shown, and Figure 5B The results for construct 5719 in the presence of TGFβ are shown. Figure 5C The percentage of tumor cell viability in construct 5718 in the absence of TGFβ is shown, and Figure 5D The results for construct 5718 in the presence of TGFβ are shown.

[0017] Figures 6A-6D This study demonstrates the in vitro re-attack of four anti-CD70 CAR constructs, either alone, with SKO, or with DKO IEE editing, against the 768-O tumor cell line, based on tumor cell area (mm²). 2 The construct was measured against the baseline construct 4645 and the standalone TRAC KO. Figure 6A The results of constructor 5719 are shown. Figure 6B The results of constructor 5281 are shown. Figure 6C The results for constructor 5715 are shown, and Figure 6D The results of constructor 6115 are displayed.

[0018] Figures 7A-7DFour anti-CD70 CAR constructs, either alone, with SKO, or with DKO IEE editing, were shown to demonstrate in vitro re-attack against ACHN tumor cell lines, based on tumor cell area (mm²). 2 The construct was measured against the baseline construct 4645 and the standalone TRAC KO. Figure 7A The results of constructor 5719 are shown. Figure 7B The results of constructor 5281 are shown. Figure 7C The results for constructor 5715 are shown, and Figure 7D The results of constructor 6115 are displayed.

[0019] Figures 8A-8C The efficacy of three anti-CD70 CAR constructs, individually or with SKO or DKO IEE editing, relative to the baseline construct 4645 was demonstrated in a 786-O mouse tumor cell model, as a function of tumor volume (mm²). 3 To measure. Figure 8A The results of constructor 5719 are shown. Figure 8B The results for constructor 5715 are shown, and Figure 8C The results of constructor 5281 are displayed.

[0020] Figure 9A-9G Shown in Figure 8A -C The result of re-attack on anti-CD70CAR constructs with SKO or DKO IEE editing that completely control tumor growth, which is determined by tumor volume (mm). 3 The construct was measured using [method name missing]. It was compared to mice with only tumors. Figure 9A The results of the re-attack on construct 5719 + CD70 KO are shown. Figure 9B The results of the re-attack on construct 5719 + TGFβR2 KO are shown. Figure 9C The results of the re-attack on the construct 5715 + CD70 + TGFβR2 DKO are shown. Figure 9D The results of the re-attack on construct 5281 + TGFβR2 KO are shown. Figure 9E The results of the re-attack on construct 5281 + CD70 + TGFβR2DKO are shown. Figure 9F The results of the re-attack on the construct 5719 + CD70 + TGFβR2 DKO are shown. Figure 9G The results of the re-attack on construct 5715 + TGFβR2 KO are shown.

[0021] Figure 10The percentage of allogeneic CD70 CAR-T cell editing per edit is shown for each of the three donors, as assessed by flow cytometry or genome sequencing (results from each donor are shown as solid dots).

[0022] Figure 11A-11C The percentage of CAR T cells presenting the specified activation markers is displayed. Figure 11A The percentage of CAR T cells that are positive for CD69 is shown. Figure 11B The percentage of CAR T cells that are positive for CD107a is shown, and Figure 11C The percentage of CAR T cells that are positive for CD25 is shown.

[0023] Figure 12A-12B The results of re-attacks with three different batches of CAR-T cells against tumor cell lines with high and moderate CD70 expression are shown, as measured by the number of tumor cells. Figure 12A The results of re-attack with T cells targeting the 786-O tumor cell line were shown, and Figure 12B The results of re-attack with T cells targeting the ACHN tumor cell line are shown.

[0024] Figure 13 This study demonstrates the efficacy of two different batches of T cells against 786-O tumor cells at three different doses (10e6, 3e6, 1e6) over a 115-day period, as shown by changes in tumor volume (mm). 3 ) was measured.

[0025] Figure 14 The study demonstrated the efficacy of engineered T cells against 11 different PDX tumor models over a 42-day period, as well as the effect of tumor volume (mm²) on tumor size. 3 ) was measured.

[0026] Figure 15 This section displays karyotype analysis data comparing edited cells with donor-matched unedited controls. Two hundred cell spreads were analyzed for each sample (N = 3 donors). Statistical analysis for each indicator aberration was performed donor-by-donor using Fisher's Exact Test. * indicates p < 0.05 for any donor group. Bars represent the mean + / - SD from three matched donors (points).

[0027] Figures 16A-16BThe average percentage of host NK cells, normalized for individual CAR groups, killed engineered donor T cells (all donors in the B2M CD70-CAR T cell group or all donors in the allogeneic CD70-CAR T cell group) after treatment with host NK cells that were either genotype mismatched or HLA-C matched. Figure 16A The results of the genotype mismatch system were shown, and Figure 16B The results of the HLA-C matching system are shown.

[0028] Figures 17A-17C The average percentage of proliferation of engineered donor T cells (all donors in the CAR group alone (solid circles) or all donors in the allogeneic CD70 CAR T cell group (solid squares)) compared to normalized values ​​after treatment with host PBMCs that were either genotype mismatched or C-matched. Figure 17A The results of the genotype mismatch system were shown, and Figure 17B The results of the C matching system are displayed. Figure 17C The average percentage of proliferation of donor T cells engineered in the presence of autologous PBMCs is shown. Detailed Implementation

[0029] This disclosure provides chimeric antigen receptors (CARs) and methods and compositions for manufacturing CAR-containing immune effector cells (e.g., T cells and NK cells). In embodiments, the cells are T cells, which are engineered to express CARs, for example, as described herein. In embodiments, the CAR is an anti-CD70 CAR, for example, as described herein.

[0030] In some embodiments, the disclosure provided herein further relates to the use of CAR technology in conjunction with genome editing using a CRISPR / Cas system (e.g., the Cas9 system). In some embodiments, this disclosure describes genetically modified CAR-transduced cells, as well as gRNA molecules, compositions, and methods for using genetically modified CAR-transduced cells. In particular, the gRNA molecules, compositions, and methods described herein relate to regulating the expression (or the expression of a functional form thereof) of target molecules that affect the function of transplanted cells (e.g., cells used for cancer immunotherapy). In one embodiment, the transplanted cells are immune effector cells, such as NK cells or T cells. In one embodiment, the cells are allogeneic cells. Therefore, this document provides compositions and methods for altering (e.g., inhibiting or reducing) the expression and / or function (e.g., expression level of functional types) of a gene target or a protein encoded by a gene target, thereby improving the efficacy (e.g., transplanted immune effector cells, such as NK cells or T cells, such as T cells engineered to express CARs, such as allogeneic CAR-expressing T cells for immunotherapy), function, proliferation, stimulation, or survival.

[0031] In some implementations, the gene target is an allogeneic T-cell protein, such as HLA-A, HLA-B, TRAC, or CIITA. Without being bound by theory, it is believed that inhibiting or eliminating the level of allogeneic T-cell targets or the expression level of allogeneic T-cell target gene targets (e.g., via gene alteration) can improve the function of cells, such as transplanted cells, transplanted immune effector cells, such as CAR-T cells, such as allogeneic CAR-T cells, or will induce resistance of said transplanted cells to immunosuppressive therapy.

[0032] In one aspect, the compositions and methods described herein can be used to improve the function (e.g., by reducing or eliminating undesirable immunogenicity (such as host-graft resistance or graft-versus-host disease)), survival, proliferation, and / or efficacy of cells, such as T cells, such as CAR-engineered T cells, such as allogeneic CAR-engineered T cells, by altering the genes of components of the major histocompatibility complex (e.g., HLA proteins, such as HLA-A and / or HLA-B). While not wishing to be bound by theory, it is believed that the reduction or absence of expression of mismatched (e.g., mismatched with the type of subject receiving cell therapy) HLA proteins (or components) reduces or eliminates host-versus-graft disease by eliminating host T cell receptor recognition and response to mismatched (e.g., allogeneic) transplanted tissues. Therefore, this approach can be used to generate “off-the-shelf” T cells (Torikai et al., 2012 Blood 119, 5697-5705).

[0033] In one aspect, the compositions and methods described herein can be used to improve the function (e.g., by reducing or eliminating unwanted immunogenicity (such as host resistance to graft-versus-host disease)), survival, proliferation, and / or efficacy of cells, such as T cells, CAR-engineered T cells, and allogeneic CAR-engineered T cells, by altering the genes of components of T cell receptors (TCRs), such as TRACs. While not wishing to be bound by theory, it is thought that reduced or absent expression of functional T cell receptor components would reduce or eliminate the presence of TCRs on the surface of said cells, thereby reducing or preventing graft-versus-host disease by eliminating T cell receptor recognition and response to host tissues. Therefore, this method can be used to generate “off-the-shelf” T cells.

[0034] In one aspect, the compositions and methods described herein can be used to improve the function (e.g., by reducing or eliminating undesirable immunogenicity (such as host resistance to graft-versus-host disease)), survival, proliferation, and / or efficacy of cells, such as T cells, such as CAR-engineered T cells, such as allogeneic CAR-engineered T cells, by altering genes (e.g., CIITA) encoding proteins that regulate the expression of one or more components of the major histocompatibility complex. While not wishing to be bound by theory, it is believed that reducing or eliminating the expression of regulators of MHC class II expression (e.g., CIITA) will reduce or eliminate the expression of MHC class II molecules on allogeneic cells, thereby reducing or eliminating the expression of mismatched (e.g., mismatched with the type of subject receiving cell therapy) MHC class II proteins (or components), thereby reducing or eliminating host resistance to graft-versus-host disease, as described herein, by, for example, eliminating host T cell receptor recognition and response to mismatched (e.g., allogeneic) transplanted tissues (e.g., allogeneic T cells, such as allogeneic CAR-T cells). Therefore, this method can be used to generate "ready-made" T cells.

[0035] In one embodiment, it may be advantageous to reduce or eliminate the expression of one or more MHC class I molecules and one or more MHC class II molecules, for example, in T cells, such as allogeneic T cells, such as allogeneic CAR-T cells, for example, as described herein, to further reduce or eliminate host resistance to graft-versus-graft disease after administration of the cells. Therefore, in embodiments of the cells and methods of this disclosure, cells may be contacted with compositions of this disclosure (e.g., compositions comprising gRNA and Cas9 molecules) containing gRNA molecules targeting HLA-A or HLA-B (e.g., as described herein) (e.g., to reduce or eliminate the expression of one or more MHC class I molecules in the cells), and with compositions of this disclosure (e.g., compositions comprising gRNA and Cas9 molecules) containing gRNA molecules targeting CIITA (e.g., as described herein) (e.g., to reduce or eliminate the expression of one or more MHC class II molecules). In embodiments of the cells and methods disclosed herein, cells may also be contacted with compositions of this disclosure (e.g., compositions comprising gRNA and Cas9 molecules) containing gRNA molecules (e.g., as described herein) that contain a component targeting the TCR (e.g., targeting TRAC). This contact may reduce or eliminate the expression of T cell receptors (e.g., one or more components of the TCR). In one embodiment, the cells of this disclosure have reduced or eliminated TCR expression (e.g., as detected by flow cytometry), reduced or eliminated expression of one or more MHC class I molecules (e.g., as detected by flow cytometry), and reduced or eliminated expression of one or more MHC class II molecules (e.g., as detected by flow cytometry).

[0036] In embodiments, reduced or eliminated expression is measured relative to similar cells that have not been treated with the compositions or CRISPR systems of this disclosure. In embodiments, the cells are immune effector cells, such as T cells or NK cells, such as T cells, as described herein. In embodiments, the cells are human cells. In embodiments, the cells are allogeneic relative to the subject to whom the cells are to be administered. In embodiments, reduced or eliminated HLA-A, HLA-B, TRAC, and / or CIITA expression is achieved by introducing the compositions, CRISPR systems, or gRNAs of this disclosure into the cells, for example, as described herein, or by methods as described herein.

[0037] In another embodiment, the compositions and methods described herein can be used to improve the function (e.g., by reducing or eliminating undesirable immunogenicity (such as host-to-graft or graft-to-host response)), survival, proliferation, and / or efficacy of cells, such as T cells, CAR-transduced T cells, or allogeneic CAR-transduced T cells, by altering the genes of immune signaling proteins (e.g., TGFBR2 or CD70). Thus, this method can be used to generate “off-the-shelf” T cells.

[0038] In embodiments, reduced or eliminated expression is measured relative to similar cells that have not been treated with the compositions or CRISPR systems of this disclosure. In embodiments, the cells are immune effector cells, such as T cells or NK cells, such as T cells, as described herein. In embodiments, the cells are human cells. In embodiments, the cells are allogeneic relative to the subject to whom the cells are to be administered. In embodiments, reduced or eliminated TGFBR2 or CD70 expression is achieved by introducing the compositions, CRISPR systems, or gRNA of this disclosure into the cells, for example, as described herein, or by methods as described herein.

[0039] In embodiments where cells have reduced or eliminated HLA-A levels or expression levels, the cells contain gene modifications within genomic coordinates targeted by guide RNA, said guide RNA comprising a guide sequence of any one of SEQ ID NO: 403, 404, and 412, or a gRNA molecule comprising a guide sequence of 20, 21, 22, 23, 24, or 25 consecutive nucleotides, preferably 20 consecutive nucleotides, of any one of SEQ ID NO: 403, 404, and 412.

[0040] In embodiments where cells have reduced or eliminated HLA-B levels or expression levels, the cells contain gene modifications within genomic coordinates targeted by guide RNA, said guide RNA comprising a guide sequence of any of SEQ ID NO: 405-407, or a gRNA molecule comprising a guide sequence of 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides, preferably 20 consecutive nucleotides, of any of SEQ ID NO: 405-407.

[0041] In embodiments where cells have reduced or eliminated TRAC levels or expression levels, the cells contain gene modifications within genomic coordinates targeted by guide RNA, the guide RNA comprising a guide sequence of SEQ ID NO: 413, or a gRNA molecule comprising a guide sequence of 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides, preferably 20 consecutive nucleotides, of SEQ ID NO: 413.

[0042] In embodiments where cells have reduced or eliminated CIITA levels or expression levels, the cells contain gene modifications within genomic coordinates targeted by the guide RNA, said guide RNA comprising a guide sequence of any one of SEQ ID NO: 401, 402, and 411, or a gRNA molecule comprising a guide sequence of 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides, preferably 20 consecutive nucleotides, of any one of SEQ ID NO: 401, 402, and 411.

[0043] In embodiments where cells have reduced or eliminated TGFBR2 levels or expression levels, the cells contain gene modifications within genomic coordinates targeted by guide RNA, said guide RNA comprising a guide sequence of any of SEQ ID NO: 301-309, 371 or 372, or a gRNA molecule comprising a guide sequence of 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides, preferably 20 consecutive nucleotides, of any of SEQ ID NO: 301-309, 371 or 372.

[0044] In embodiments where cells have reduced or eliminated CD70 levels or expression levels, the cells contain gene modifications within genomic coordinates targeted by the guide RNA, said guide RNA comprising a guide sequence of any of SEQ ID NO: 310-319, or a gRNA molecule comprising a guide sequence of 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides, preferably 20 consecutive nucleotides, of any of SEQ ID NO: 310-319.

[0045] The terms “about” or “approximately” mean an acceptable error (in part depending on how the value is measured or determined) in relation to a particular value as determined by a person skilled in the art, or a degree of variation that does not substantially affect the characteristics of the subject matter or is within tolerances acceptable in the art (e.g., within 10%, 5%, 2%, or 1%). Therefore, unless the contrary is indicated, the numerical parameters set forth in the following description and the appended claims are approximations that may vary depending on the desired characteristics sought to be obtained. To a minimum, and not at all, to limit the application of the doctrine of equivalence to the scope of the claims, each numerical parameter should be interpreted based on at least the number of significant digits reported and by applying common rounding techniques.

[0046] The following implementation schemes are provided. Additional implementation schemes are provided throughout this disclosure.

[0047] Implementation Scheme 1 is an anti-CD70 chimeric antigen receptor (CAR) comprising: a. an antigen-binding protein or fragment thereof that specifically binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region including complementarity-determining region 1 (VH CDR1), VH CDR2, and VH CDR3, and a light chain variable (VL) region including complementarity-determining region 1 (VL CDR1), VL CDR2, and VL CDR3, and wherein i. the VH CDR1 comprises the amino acid sequence of any one of SEQ ID NO: 67, 70, 73, 76, 79, and 82; ii. the VH CDR2 comprises the amino acid sequence of any one of SEQ ID NO: 68, 71, 74, 77, 80, and 83; iii. the VH CDR3 comprises the amino acid sequence of any one of SEQ ID NO: 69, 72, 75, 78, 81, and 84; iv. the VL CDR1 comprises the amino acid sequence of any one of SEQ ID NO: a. an amino acid sequence of any one of SEQ ID NO: 49, 52, 55, 58, 61, and 64; b. the VL CDR2 comprising an amino acid sequence of any one of SEQ ID NO: 50, 53, 56, 59, 62, and 65; and c. the VL CDR3 comprising an amino acid sequence of any one of SEQ ID NO: 51, 54, 57, 60, 63, and 66; d. a transmembrane domain; and e. an intracellular domain comprising a costimulatory domain comprising an amino acid sequence of SEQ ID NO: 99 or 101.

[0048] Implementation scheme 1.1 is an anti-CD70 CAR as described in implementation scheme 1, wherein the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2 and the VL CDR3 comprise the following amino acid sequences: (a) SEQ ID NO: 73, 74, 75, 55, 56 and 57, respectively; (b) SEQ ID NO: 67, 68, 69, 49, 50 and 51, respectively; (c) SEQ ID NO: 70, 71, 72, 52, 53 and 54, respectively; (d) SEQ ID NO: 76, 77, 78, 58, 59 and 60, respectively; (e) SEQ ID NO: 79, 80, 81, 61, 62 and 63, respectively; or (f) SEQ ID NO: 82, 83, 84, 64, 65 and 66, respectively.

[0049] Implementation scheme 1.2 is an anti-CD70 CAR as described in implementation scheme 1 or 2, wherein the VH CDR1, the VHCDR2, the VH CDR3, the VL CDR1, the VL CDR2 and the VL CDR3 contain the following amino acid sequences: SEQ ID NO: 73, 74, 75, 55, 56 and 57, respectively.

[0050] Implementation scheme 2 is an anti-CD70 CAR as described in any one of implementation schemes 1, 1.1 and 1.2, wherein the VH region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with any of SEQ ID NO: 43-48.

[0051] Implementation scheme 3 is an anti-CD70 CAR as described in any one of implementation schemes 1, 1.1, 1.2 and 2, wherein the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with any of SEQ ID NO: 37-42.

[0052] Implementation scheme 3.1 is an anti-CD70 CAR as described in any one of implementation schemes 1, 1.1, 1.2, 2, and 3, wherein: (a) the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39; (b) the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 43, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 37; (c) the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 37. 44 has an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 38, and the VL region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 38; (d) the VH region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 46, and the VL region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 40; (e) the VH region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 47, and the VL region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 47. 41 has an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 48; or (f) the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 42, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 42.

[0053] Implementation scheme 3.2 is an anti-CD70 CAR as described in any one of implementation schemes 1, 1.1, 1.2, 2, 3 and 3.1, wherein: the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 45, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 39.

[0054] Implementation scheme 3.3 is an anti-CD70CAR as described in any one of implementation schemes 1, 1.1, 1.2, 2, 3, 3.1, and 3.2, wherein: (a) the VH region contains the amino acid sequence of SEQ ID NO: 45, and the VL region contains the amino acid sequence of SEQ ID NO: 39; (b) the VH region contains the amino acid sequence of SEQ ID NO: 43, and the VL region contains the amino acid sequence of SEQ ID NO: 37; (c) the VH region contains the amino acid sequence of SEQ ID NO: 44, and the VL region contains the amino acid sequence of SEQ ID NO: 38; (d) the VH region contains the amino acid sequence of SEQ ID NO: 46, and the VL region contains the amino acid sequence of SEQ ID NO: 40; (e) the VH region contains the amino acid sequence of SEQ ID NO: 47, and the VL region contains the amino acid sequence of SEQ ID NO: 41; or (f) the VH region contains the amino acid sequence of SEQ ID NO: 48, and the VL region contains the amino acid sequence of SEQ ID NO: 49. The amino acid sequence of NO: 42.

[0055] Implementation scheme 3.4 is an anti-CD70CAR as described in any one of implementation schemes 1, 1.1, 1.2, 2, 3 and 3.1-3.3, wherein: the VH region contains the amino acid sequence of SEQ ID NO: 45, and the VL region contains the amino acid sequence of SEQ ID NO: 39.

[0056] Implementation scheme 4 is an anti-CD70 CAR as described in any one of implementation schemes 1-3, 1.1, 1.2 and 3.1-3.4, wherein the antigen-binding protein comprises the amino acid sequence of any one of SEQ ID NO: 25, 27, 29, 32, 34 and 36, or a sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO: 25, 27, 29, 32, 34 and 36.

[0057] Embodiment 5 is an anti-CD70 CAR as described in any one of Embodiments 1-4, 1.1, 1.2 and 3.1-3.4, wherein the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25.

[0058] Embodiment 6 is an anti-CD70 CAR as described in any one of Embodiments 1-4, 1.1, 1.2 and 3.1-3.4, wherein the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 27.

[0059] Embodiment 7 is an anti-CD70 CAR as described in any one of Embodiments 1-4, 1.1, 1.2 and 3.1-3.4, wherein the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 27, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 99.

[0060] Embodiment 8 is an anti-CD70 CAR as described in any one of Embodiments 1-4, 1.1, 1.2 and 3.1-3.4, wherein the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 27, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 101.

[0061] Embodiment 9 is an anti-CD70 CAR as described in any one of Embodiments 1-8, 1.1, 1.2 and 3.1-3.4, wherein the transmembrane domain comprises a CD8a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 95.

[0062] Embodiment 10 is an anti-CD70 CAR as described in any one of Embodiments 1-8, 1.1, 1.2 and 3.1-3.4, wherein the transmembrane domain comprises a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 93.

[0063] Implementation scheme 11 is an anti-CD70 CAR as described in any one of implementation schemes 1-10, 1.1, 1.2 and 3.1-3.4, wherein the anti-CD70 CAR further comprises a hinge domain between the antigen-binding protein and the transmembrane domain.

[0064] Embodiment 12 is an anti-CD70 CAR as in Embodiment 11, wherein the hinge domain is a CD8a hinge domain or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 89.

[0065] Implementation scheme 13 is an anti-CD70 CAR as described in any one of implementation schemes 1-12, 1.1, 1.2 and 3.1-3.4, wherein the intracellular domain further comprises an activation domain.

[0066] Embodiment 14 is an anti-CD70 CAR as described in Embodiment 13, wherein the activation domain is a CD3z activation domain comprising the amino acid sequence of SEQ ID NO: 103.

[0067] Embodiment 15 is an anti-CD70 CAR as described in any one of Embodiments 1-14, 1.1, 1.2 and 3.1-3.4, wherein the intracellular domain comprises a CD28 co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 99 and a CD3z activation domain comprising the amino acid sequence of SEQ ID NO: 103.

[0068] Embodiment 16 is an anti-CD70 CAR as described in any one of Embodiments 1-15, 1.1, 1.2 and 3.1-3.4, wherein the intracellular domain comprises a 41BB co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 101 and a CD3z activation domain comprising the amino acid sequence of SEQ ID NO: 103.

[0069] Embodiment 17 is an anti-CD70 CAR as described in any one of Embodiments 1-16, wherein: a. the antigen-binding protein comprises the sequence of SEQ ID NO: 32 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; b. the antigen-binding protein comprises the sequence of SEQ ID NO: 32 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; c. the antigen-binding protein comprises the sequence of SEQ ID NO: 25 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; d. the antigen-binding protein comprises the sequence of SEQ ID NO: 25 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; e. the antigen-binding protein comprises the sequence of SEQ ID NO: 27 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; f. the antigen-binding protein comprises the sequence of SEQ ID NO: 27 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; g. the antigen-binding protein comprises the sequence of SEQ ID NO: 36 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101. The sequence of NO: 101; or h. the antigen-binding protein comprises the sequence of SEQ ID NO: 36 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99.

[0070] Embodiment 18 is an anti-CD70 CAR as described in any one of Embodiments 1-17, 1.1, 1.2 and 3.1-3.4, wherein the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 14, 16, 20 and 22.

[0071] Embodiment 19 is an anti-CD70 CAR as described in any one of Embodiments 1-18, 1.1, 1.2 and 3.1-3.4, wherein the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 4.

[0072] Embodiment 20 is an anti-CD70 CAR as described in any one of Embodiments 1-19, 1.1, 1.2 and 3.1-3.4, wherein the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 8.

[0073] Embodiment 21 is an anti-CD70 CAR as described in any one of Embodiments 1-20, 1.1, 1.2 and 3.1-3.4, wherein the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 2.

[0074] Implementation scheme 22 is an anti-CD70 CAR as described in any one of implementation schemes 1-21, 1.1, 1.2 and 3.1-3.4, wherein the antigen-binding protein is scFv.

[0075] Implementation scheme 23 is an anti-CD70 CAR as described in implementation scheme 22, wherein the scFv includes a connector between the VH region and the VL region.

[0076] Embodiment 24 is an anti-CD70 CAR as described in Embodiment 22 or 23, wherein the scFv comprises a glycine-serine linker between the VH region and the VL region, optionally the glycine-serine linker comprising the sequence of SEQ ID NO: 940.

[0077] Implementation scheme 25 is a nucleic acid that encodes an anti-CD70 CAR as described in any one of implementation schemes 1-24, 1.1, 1.2 and 3.1-3.4.

[0078] Implementation scheme 26 is a nucleic acid as described in implementation scheme 25, said nucleic acid comprising a nucleic acid sequence of any one of SEQ ID NO: 23, 24, 26, 28, 30, 31, 33 and 35, or a nucleic acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO: 23, 24, 26, 28, 30, 31, 33 and 35.

[0079] Implementation scheme 27 is a nucleic acid as described in implementation scheme 25 or 26, said nucleic acid comprising a nucleic acid sequence of any one of SEQ ID NO: 96-98 and 100, or a nucleic acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO: 96-98 and 100.

[0080] Implementation scheme 28 is a nucleic acid as described in any one of implementation schemes 25-27, wherein the anti-CD70 CAR comprises a hinge domain, wherein the nucleic acid comprises a nucleic acid encoding the hinge domain, and the hinge domain comprises a nucleic acid sequence of any one of SEQ ID NO: 85-88, or a nucleic acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 85-88.

[0081] Embodiment 29 is a nucleic acid as described in any one of Embodiments 25-28, said nucleic acid comprising a nucleic acid sequence encoding the transmembrane domain, said transmembrane domain comprising a sequence of any one of SEQ ID NO: 90-92 and 94, or a sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 90-92 and 94.

[0082] Implementation scheme 30 is a nucleic acid as described in any one of implementation schemes 25-29, wherein the intracellular domain comprises an activation domain, wherein the nucleic acid comprises a nucleic acid encoding the activation domain, and the activation domain comprises a nucleic acid sequence of SEQ ID NO: 102 or 104, or a nucleic acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 102 or 104.

[0083] Implementation scheme 31 is a nucleic acid as described in any one of implementation schemes 25-30, said nucleic acid comprising a nucleic acid sequence of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21, or a nucleic acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21.

[0084] Implementation scheme 32 is a nucleic acid as described in any one of implementation schemes 25-31, said nucleic acid comprising the nucleic acid sequence of any one of SEQ ID NO: 1, 3 and 7.

[0085] Implementation scheme 33 is an mRNA encoded by a nucleic acid as described in any one of implementation schemes 25-32.

[0086] Implementation scheme 34 is an expression vector that is operatively linked to or contains a nucleic acid as described in any one of implementation schemes 25-32.

[0087] Implementation scheme 35 is an engineered cell that comprises a nucleic acid as described in any one of implementation schemes 25-32, an mRNA as described in implementation scheme 33, or an expression vector as described in implementation scheme 34.

[0088] Implementation scheme 36 is an engineered cell comprising an anti-CD70 CAR as described in any one of implementation schemes 1-24, 1.1, 1.2 and 3.1-3.4.

[0089] Embodiment 37 is an engineered cell comprising an anti-CD70 CAR as described in any one of Embodiments 1-24, 1.1, 1.2 and 3.1-3.4, wherein the cell is transduced with an expression vector operatively linked to or comprising a nucleic acid encoding the anti-CD70 CAR, and wherein the expression vector directs the expression of the anti-CD70 CAR in the cell.

[0090] Embodiment 38 is an expression vector as described in Embodiment 34 or an engineered cell as described in Embodiment 35 or 37, wherein the expression vector comprises a retroviral or lentiviral expression vector.

[0091] Embodiment 39 is an expression vector as described in Embodiment 34 or an engineered cell as described in Embodiment 35 or 37, wherein the expression vector comprises an AAV vector.

[0092] Embodiment 40 is an expression vector or engineered cell as described in Embodiment 39, wherein the expression vector comprises SEQ ID NO: 106.

[0093] Implementation scheme 41 is an engineered cell as described in implementation scheme 40, wherein the engineered cell comprises SEQ ID NO: 107.

[0094] Implementation Scheme 42 is an engineered cell comprising an anti-CD70 chimeric antigen receptor (CAR), wherein the anti-CD70 CAR comprises: a. an antigen-binding protein or fragment thereof that specifically binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region comprising complementarity-determining region 1 (VH CDR1), VH CDR2, and VH CDR3, and a light chain variable (VL) region comprising complementarity-determining region 1 (VL CDR1), VL CDR2, and VL CDR3, and wherein i. the VH CDR1 comprises the amino acid sequence of any one of SEQ ID NO: 67, 70, 73, 76, 79, and 82; ii. the VH CDR2 comprises the amino acid sequence of any one of SEQ ID NO: 68, 71, 74, 77, 80, and 83; iii. the VH CDR3 comprises the amino acid sequence of any one of SEQ ID NO: 69, 72, 75, 78, 81, and 84; iv. the VL CDR1 comprises the amino acid sequence of any one of SEQ ID NO: a. an amino acid sequence of any one of SEQ ID NO: 49, 52, 55, 58, 61, and 64; b. the VL CDR2 comprising an amino acid sequence of any one of SEQ ID NO: 50, 53, 56, 59, 62, and 65; c. the VL CDR3 comprising an amino acid sequence of any one of SEQ ID NO: 51, 54, 57, 60, 63, and 66; d. a transmembrane domain; and e. an intracellular domain comprising a costimulatory domain comprising an amino acid sequence of SEQ ID NO: 99 or 101.

[0095] Implementation scheme 42.1 is an engineered cell as described in implementation scheme 42, wherein the VH CDR1, VHCDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the following amino acid sequences: (a) SEQ ID NO: 73, 74, 75, 55, 56 and 57, respectively; (b) SEQ ID NO: 67, 68, 69, 49, 50 and 51, respectively; (c) SEQ ID NO: 70, 71, 72, 52, 53 and 54, respectively; (d) SEQ ID NO: 76, 77, 78, 58, 59 and 60, respectively; (e) SEQ ID NO: 79, 80, 81, 61, 62 and 63, respectively; or (f) SEQ ID NO: 82, 83, 84, 64, 65 and 66, respectively.

[0096] Implementation scheme 42.2 is an engineered cell as described in implementation scheme 42 or 42.1, wherein the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2 and the VL CDR3 contain the following amino acid sequences: SEQ ID NO: 73, 74, 75, 55, 56 and 57, respectively.

[0097] Embodiment 43 is an engineered cell as described in any one of Embodiments 42, 42.1 and 42.2, wherein the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO: 43-48.

[0098] Embodiment 44 is an engineered cell as described in any one of embodiments 42, 42.1, 42.2 and 43, wherein the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO: 37-42.

[0099] Implementation scheme 44.1 is an engineered cell as described in any one of implementation schemes 42, 42.1, 42.2, 43, and 44, wherein: (a) the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39; (b) the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 43, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 37; (c) the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 49. 44 has an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 38, and the VL region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 48; (d) the VH region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 46, and the VL region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 40; (e) the VH region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 47, and the VL region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 48. 41 has an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 48; or (f) the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 42, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 42.

[0100] Implementation scheme 44.2 is an engineered cell as described in any one of implementation schemes 42, 42.1, 42.2, 43, 44 and 44.1, wherein: the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 45, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 39.

[0101] Implementation scheme 44.3 is an engineered cell as described in any one of implementation schemes 42, 42.1, 42.2, 43, 44, 44.1, and 44.2, wherein: (a) the VH region contains the amino acid sequence of SEQ ID NO: 45, and the VL region contains the amino acid sequence of SEQ ID NO: 39; (b) the VH region contains the amino acid sequence of SEQ ID NO: 43, and the VL region contains the amino acid sequence of SEQ ID NO: 37; (c) the VH region contains the amino acid sequence of SEQ ID NO: 44, and the VL region contains the amino acid sequence of SEQ ID NO: 38; (d) the VH region contains the amino acid sequence of SEQ ID NO: 46, and the VL region contains the amino acid sequence of SEQ ID NO: 40; (e) the VH region contains the amino acid sequence of SEQ ID NO: 47, and the VL region contains the amino acid sequence of SEQ ID NO: 41; or (f) the VH region contains the amino acid sequence of SEQ ID NO: 49. The amino acid sequence of SEQ ID NO: 48, and the VL region contains the amino acid sequence of SEQ ID NO: 42.

[0102] Implementation scheme 44.4 is an engineered cell as described in any one of implementation schemes 42, 42.1, 42.2, 43, 44 and 44.1-44.3, wherein: the VH region contains the amino acid sequence of SEQ ID NO: 45, and the VL region contains the amino acid sequence of SEQ ID NO: 39.

[0103] Embodiment 45 is an engineered cell as described in any one of Embodiments 42-44, 42.1, 42.2 and 44.1-44.4, wherein the antigen-binding protein comprises the amino acid sequence of any one of SEQ ID NO: 25, 27, 29, 32, 34 and 36, or a sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO: 25, 27, 29, 32, 34 and 36.

[0104] Embodiment 46 is an engineered cell as described in any one of embodiments 42-45, 42.1, 42.2 and 44.1-44.4, wherein the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25.

[0105] Embodiment 47 is an engineered cell as described in any one of Embodiments 42-45, 42.1, 42.2 and 44.1-44.4, wherein the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 27.

[0106] Embodiment 48 is an engineered cell as described in any one of Embodiments 42-47, 42.1, 42.2 and 44.1-44.4, wherein the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 27, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 99.

[0107] Embodiment 49 is an engineered cell as described in any one of Embodiments 42-47, 42.1, 42.2 and 44.1-44.4, wherein the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 27, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 101.

[0108] Embodiment 50 is an engineered cell as described in any one of Embodiments 42-49, 42.1, 42.2 and 44.1-44.4, wherein the transmembrane domain comprises a CD8a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 95.

[0109] Embodiment 51 is an engineered cell as described in any one of embodiments 42-49, 42.1, 42.2 and 44.1-44.4, wherein the transmembrane domain comprises a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 93.

[0110] Embodiment 52 is an engineered cell as described in any one of Embodiments 42-51, 42.1, 42.2 and 44.1-44.4, wherein the engineered cell further comprises a hinge domain between the antigen-binding protein and the transmembrane domain.

[0111] Embodiment 53 is an engineered cell as described in any one of embodiments 42-52, 42.1, 42.2 and 44.1-44.4, wherein the hinge domain is a CD8a hinge domain or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 89.

[0112] Implementation scheme 54 is an engineered cell as described in any one of implementation schemes 42-53, 42.1, 42.2 and 44.1-44.4, wherein the intracellular domain further comprises an activation domain.

[0113] Embodiment 55 is an engineered cell as described in any one of embodiments 42-54, 42.1, 42.2 and 44.1-44.4, wherein the activation domain is a CD3z activation domain comprising the amino acid sequence of SEQ ID NO: 103.

[0114] Embodiment 56 is an engineered cell as described in any one of Embodiments 42-55, 42.1, 42.2 and 44.1-44.4, wherein the intracellular domain comprises a CD28 co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 99 and a CD3z activation domain comprising the amino acid sequence of SEQ ID NO: 103.

[0115] Embodiment 57 is an engineered cell as described in any one of Embodiments 42-55, 42.1, 42.2 and 44.1-44.4, wherein the intracellular domain comprises a 41BB co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 101 and a CD3z activation domain comprising the amino acid sequence of SEQ ID NO: 103.

[0116] Embodiment 58 is an engineered cell as described in any one of Embodiments 42-57, 42.1, 42.2, and 44.1-44.4, wherein: a. the antigen-binding protein comprises the sequence of SEQ ID NO: 32 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; b. the antigen-binding protein comprises the sequence of SEQ ID NO: 32 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; c. the antigen-binding protein comprises the sequence of SEQ ID NO: 25 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; d. the antigen-binding protein comprises the sequence of SEQ ID NO: 25 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; e. the antigen-binding protein comprises the sequence of SEQ ID NO: 27 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; f. the antigen-binding protein comprises the sequence of SEQ ID NO: 27 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101. g. The antigen-binding protein comprises the sequence of SEQ ID NO: 36 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; or h. The antigen-binding protein comprises the sequence of SEQ ID NO: 36 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99.

[0117] Embodiment 59 is an engineered cell as described in any one of Embodiments 42-58, 42.1, 42.2 and 44.1-44.4, wherein the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequences of SEQ ID NO: 2, 4, 6, 8, 14, 16, 20 and 22.

[0118] Embodiment 60 is an engineered cell as described in any one of Embodiments 42-59, 42.1, 42.2 and 44.1-44.4, wherein the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 4.

[0119] Embodiment 61 is an engineered cell as described in any one of Embodiments 42-59, 42.1, 42.2 and 44.1-44.4, wherein the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 8.

[0120] Embodiment 62 is an engineered cell as described in any one of Embodiments 42-59, 42.1, 42.2 and 44.1-44.4, wherein the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 2.

[0121] Implementation scheme 63 is an engineered cell as described in any one of implementation schemes 42-62, 42.1, 42.2 and 44.1-44.4, wherein the antigen-binding protein is scFv.

[0122] Implementation scheme 64 is an engineered cell as described in implementation scheme 63, wherein the scFv contains a connector between the VH region and the VL region.

[0123] Embodiment 65 is an engineered cell as described in Embodiment 63 or 64, wherein the scFv contains a glycine-serine linker between the VH region and the VL region, optionally the linker containing the sequence of SEQ ID NO: 940.

[0124] Embodiment 66 is a cell population comprising engineered cells as described in any one of embodiments 42-65, 42.1, 42.2 and 44.1-44.4.

[0125] Embodiment 67 is an engineered cell or cell population as described in any one of Embodiments 42-66, 42.1, 42.2 and 44.1-44.4, wherein the engineered cell or cell population further comprises reduced or eliminated surface expression of TGFBR2 relative to unmodified cells.

[0126] Implementation scheme 68 is an engineered cell or cell population as described in any one of implementation schemes 42-67, 42.1, 42.2 and 44.1-44.4, wherein the engineered cell or cell population further comprises gene modifications in the TGFBR2 gene. Implementation scheme 69 is an engineered cell or cell population as described in implementation scheme 68, wherein the genetic modification contains at least one nucleotide in the following genomic coordinates: chr3:30606864-30691614.

[0127] Implementation scheme 70 is an engineered cell or cell population as described in implementation scheme 68 or 69, wherein the gene modification is located within the genomic coordinates chr3:30674205-30674229.

[0128] Embodiment 71 is an engineered cell or cell population as described in any one of Embodiments 68 to 70, wherein the gene modification contains at least one nucleotide within a genomic coordinate system targeted by a TGFBR2 guide RNA containing the guide sequence of SEQ ID NO: 301.

[0129] Implementation scheme 72 is an engineered cell or cell population as described in implementation scheme 68, wherein the gene modification is located within the genomic coordinates chr3:30671941-30671961.

[0130] Embodiment 73 is an engineered cell or cell population as described in Embodiment 68 or 72, wherein the gene modification contains at least one nucleotide within a genomic coordinate system targeted by a TGFBR2 guide RNA containing the guide sequence of SEQ ID NO: 302.

[0131] Embodiment 74 is an engineered cell or cell population as described in any one of Embodiments 42-73, 42.1, 42.2 and 44.1-44.4, wherein the engineered cell or cell population further comprises reduced or eliminated CD70 surface expression relative to unmodified cells.

[0132] Implementation scheme 75 is an engineered cell or cell population as described in any one of implementation schemes 42-74, 42.1, 42.2 and 44.1-44.4, wherein the engineered cell or cell population further comprises the gene modification in the CD70 gene.

[0133] Implementation scheme 76 is an engineered cell or cell population as described in implementation scheme 75, wherein the gene modification contains at least one nucleotide within the genomic coordinates chr19:6586002-6591018.

[0134] Implementation scheme 77 is an engineered cell or cell population as described in implementation scheme 76, wherein the gene modification is located in the following genomic coordinates: chr19:6590121-6590145 or chr19:6586268-6586292.

[0135] Embodiment 78 is an engineered cell or cell population as described in Embodiment 76 or 77, wherein the gene modification contains at least one nucleotide within a genomic coordinate targeted by a CD70 guide RNA containing a guide sequence of SEQ ID NO: 310 or 311.

[0136] Implementation scheme 79 is an engineered cell or cell population as described in implementation scheme 75, wherein the gene modification is located in the following genomic coordinates: chr19:6586028-6591018.

[0137] Implementation scheme 80 is an engineered cell or cell population as described in implementation scheme 79, wherein the genetic modification contains at least one nucleotide in the following genomic coordinates: chr19:6590998-6591018 or chr19:6590991-6591011.

[0138] Embodiment 81 is an engineered cell or cell population as described in Embodiment 79 or 80, wherein the gene modification contains at least one nucleotide within a genomic coordinate targeted by a CD70 guide RNA containing a guide sequence of SEQ ID NO: 312 or 313.

[0139] Embodiment 82 is an engineered cell or cell population as described in any one of Embodiments 42-81, 42.1, 42.2 and 44.1-44.4, wherein the engineered cell or cell population further comprises reduced or eliminated HLA-A surface expression relative to unmodified cells.

[0140] Implementation scheme 83 is an engineered cell or cell population as described in any one of implementation schemes 42-82, 42.1, 42.2 and 44.1-44.4, wherein the engineered cell or cell population further comprises gene modifications in the HLA-A gene.

[0141] Implementation scheme 84 is an engineered cell or cell population as described in implementation scheme 83, wherein the genetic modification comprises at least one nucleotide in the following genomic coordinates: (a) chr6:29942854-29942913 and chr6:29943518-29943619; and (b) chr6:29942540-29945459.

[0142] Implementation scheme 85 is an engineered cell or cell population as described in implementation scheme 83 or 84, wherein the gene modification is within the genomic coordinates selected from the following: chr6:29942891-29942915; and chr6:29942609-29942633.

[0143] Embodiment 86 is an engineered cell or cell population as described in any one of Embodiments 83 to 85, wherein the gene modification comprises at least one nucleotide within a genomic coordinate targeted by an HLA-A guide RNA containing a guide sequence of SEQ ID NO: 403 or 404.

[0144] Embodiment 87 is an engineered cell or cell population as described in any one of Embodiments 42-86, 42.1, 42.2 and 44.1-44.4, wherein the engineered cell or cell population has reduced or eliminated HLA-B surface expression relative to unmodified cells.

[0145] Implementation scheme 88 is an engineered cell or cell population as described in any one of implementation schemes 42-87, 42.1, 42.2 and 44.1-44.4, wherein the engineered cell or cell population further comprises gene modifications in the HLA-B gene.

[0146] Implementation scheme 89 is an engineered cell or cell population as described in implementation scheme 88, wherein the gene modification is within the genomic coordinates selected from the following: (a) chr6:31354480-31357174 and (b) chr6:31357084-31354647.

[0147] Implementation scheme 90 is an engineered cell as described in implementation scheme 88 or 89, wherein the gene modification is within the genomic coordinates selected from the following: chr6:31355222-31355246, chr6:31355221-31355245 and chr6:31355205-31355229.

[0148] Embodiment 91 is an engineered cell or cell population as described in any one of Embodiments 88 to 90, wherein the gene modification contains at least one nucleotide within a genomic coordinate system targeted by an HLA-B guide RNA containing a guide sequence of SEQ ID NO: 406, 405 or 407.

[0149] Embodiment 92 is an engineered cell or cell population as described in any one of Embodiments 42-91, 42.1, 42.2 and 44.1-44.4, wherein the engineered cell or cell population contains reduced or eliminated TRAC surface expression relative to unmodified cells.

[0150] Implementation scheme 93 is an engineered cell or cell population as described in implementation schemes 42-92, 42.1, 42.2 and 44.1-44.4, wherein the engineered cell or cell population contains the gene modification in the TRAC gene.

[0151] Implementation scheme 94 is an engineered cell or cell population as described in implementation scheme 93, wherein the gene modification contains at least one nucleotide within the genomic coordinates chr14:22547524-22547544.

[0152] Embodiment 95 is an engineered cell or cell population as described in Embodiment 93 or 94, wherein the gene modification contains at least one nucleotide within a genomic coordinate system targeted by a TRAC guide RNA containing the guide sequence of SEQ ID NO: 413.

[0153] Embodiment 96 is an engineered cell or cell population as described in any one of Embodiments 42-95, 42.1, 42.2 and 44.1-44.4, wherein the engineered cell or cell population has reduced or eliminated MHC class II surface expression relative to unmodified cells.

[0154] Implementation scheme 97 is an engineered cell or cell population as described in any one of implementation schemes 42-96, 42.1, 42.2 and 44.1-44.4, wherein the engineered cell or cell population further comprises the gene modification in the CIITA gene.

[0155] Implementation scheme 98 is an engineered cell or cell population as described in implementation scheme 97, wherein the genetic modification is within the gene coordinates selected from the following: (a) chr16:10877363-10907788 and (b) chr16:10906515-10908136.

[0156] Implementation scheme 99 is an engineered cell or cell population as described in implementation scheme 97 or 98, wherein the gene modification contains at least one nucleotide within genomic coordinates chr16:10906643-10906667 or chr16:10907504-10907528.

[0157] Embodiment 100 is an engineered cell or cell population as described in any one of Embodiments 97 to 99, wherein the gene modification contains at least one nucleotide within a genomic coordinate targeted by a CIITA guide RNA containing a guide sequence of SEQ ID NO: 402 or 401.

[0158] Implementation 101 is an engineered cell or cell population as described in any one of Implementations 68 to 100, wherein the gene modification comprises insertion / deletion, C to T substitution, or A to G substitution within the genomic coordinates.

[0159] Embodiment 102 is an engineered cell or cell population as described in any one of embodiments 68 to 101, wherein the gene modification comprises C to T substitutions within the genomic coordinates.

[0160] Implementation scheme 103 is an engineered cell that includes gene modifications in the HLA-A gene, modified TRAC gene, gene modifications in the CIITA gene, gene modifications in the TGFBR2 gene, and / or gene modifications in the CD70 gene, wherein the engineered cell expresses an anti-CD70 chimeric antigen receptor (CAR) or contains nucleic acid encoding an anti-CD70 CAR.

[0161] Implementation scheme 104 is an engineered cell that includes gene modifications in the HLA-A gene, the HLA-B gene, the TRAC gene, the CIITA gene, the TGFBR2 gene, and / or the CD70 gene, wherein the engineered cell expresses an anti-CD70 chimeric antigen receptor (CAR) or contains nucleic acid encoding an anti-CD70 CAR.

[0162] Implementation scheme 105 is an engineered cell as described in implementation scheme 103 or 104, wherein the gene modification in TGFBR2 contains at least one nucleotide in the following genomic coordinates: chr3:30606864-30691614.

[0163] Implementation scheme 106 is an engineered cell as described in implementation scheme 103 or 104, wherein the gene modification in TGFBR2 is located in the following genomic coordinates: chr3:30606891-30691605.

[0164] Implementation scheme 107 is an engineered cell as described in any one of implementation schemes 103-106, wherein the gene modification in TGFBR2 is located within the genomic coordinates chr3:30674205-30674229.

[0165] Embodiment 108 is an engineered cell as described in any one of Embodiments 103-107, wherein the gene modification in TGFBR2 contains at least one nucleotide within a genomic coordinate targeted by a TGFBR2 guide RNA containing the guide sequence of SEQ ID NO: 301.

[0166] Implementation scheme 109 is an engineered cell as described in any one of implementation schemes 103-106, wherein the gene modification in TGFBR2 is located in the following genomic coordinates: chr3:30671941-30671961.

[0167] Embodiment 110 is an engineered cell as described in any one of Embodiments 103-106 and 109, wherein the gene modification in TGFBR2 contains at least one nucleotide within a genomic coordinate targeted by a guide RNA containing the guide sequence of SEQ ID NO: 302.

[0168] Implementation scheme 111 is an engineered cell as described in any one of implementation schemes 103-110, wherein the gene modification in CD70 is within the gene coordinates chr19:6586002-6591015.

[0169] Implementation scheme 112 is an engineered cell as described in any one of implementation schemes 103-111, wherein the gene modification in CD70 is within gene coordinates chr19:6590121-6590145 or chr19:6586268-6586292.

[0170] Embodiment 113 is an engineered cell as described in any one of Embodiments 103-112, wherein the gene modification in CD70 contains at least one nucleotide within a genomic coordinate targeted by a CD70 guide RNA containing a guide sequence of SEQ ID NO: 310 or 311.

[0171] Implementation scheme 114 is an engineered cell as described in any one of implementation schemes 103-110, wherein the gene modification in CD70 is located in the following genomic coordinates: chr19: 6586028-6591018.

[0172] Embodiment 115 is an engineered cell as described in any one of embodiments 103-110 and 114, wherein the gene modification in CD70 is located within genomic coordinates chr19:6590998-6591018 or chr19:6590991-6591011, optionally containing at least one nucleotide within genomic coordinates chr19:6590998-6591018.

[0173] Embodiment 116 is an engineered cell as described in any one of Embodiments 103-110, 114 and 115, wherein the genetic modification in CD70 contains at least one nucleotide within a genomic coordinate targeted by a CD70 guide RNA containing a guide sequence comprising SEQ ID NO: 312 or 313, optionally SEQ ID NO: 312.

[0174] Implementation scheme 117 is an engineered cell as described in any one of implementation schemes 103-116, wherein the gene modification in HLA-A is within the gene coordinates chr6: 29942540-29945459.

[0175] Implementation scheme 118 is an engineered cell as described in any one of implementation schemes 103-117, wherein the gene modification in HLA-A is within the genomic coordinates selected from the following: chr6:29942891-29942915; and chr6:29942609-29942633.

[0176] Embodiment 119 is an engineered cell as described in any one of Embodiments 103-118, wherein the genetic modification in HLA-A contains at least one nucleotide within a genomic coordinate system targeted by an HLA-A guide RNA containing a guide sequence of SEQ ID NO: 403 or 404.

[0177] Implementation scheme 120 is an engineered cell as described in any one of implementation schemes 104-119, wherein the gene modification in HLA-B contains at least one nucleotide within the gene coordinates chr6:31354480-31357174.

[0178] Implementation scheme 121 is an engineered cell as described in any one of implementation schemes 104-120, wherein the gene modification in HLA-B is located in the following genomic coordinates: chr6:31355222-31355246, chr6:31355221-31355245 or chr6:31355205-31355229.

[0179] Embodiment 122 is an engineered cell as described in any one of Embodiments 104-121, wherein the genetic modification in HLA-B contains at least one nucleotide within a genomic coordinate system targeted by an HLA-B guide RNA containing the guide sequence of SEQ ID NO: 405-407.

[0180] Implementation scheme 123 is an engineered cell as described in any one of implementation schemes 104-122, wherein the gene modification in CIITA is within the gene coordinates selected from the following: (a) chr16:10877363-10907788 and (b) chr16:10906515-10908136.

[0181] Implementation scheme 124 is an engineered cell as described in any one of implementation schemes 104-123, wherein the gene modification in CIITA is within the following gene coordinates: chr16:10906643-10906667 or chr16:10907504-10907528.

[0182] Embodiment 125 is an engineered cell as described in any one of Embodiments 103-124, wherein the genetic modification in CIITA contains at least one nucleotide within a genomic coordinate targeted by a CIITA guide RNA containing a guide sequence of SEQ ID NO: 402 or 401.

[0183] Implementation scheme 126 is an engineered cell as described in any one of implementation schemes 103-125, wherein the gene modification in the TRAC is located within the genomic coordinates chr14:22547524-22547544.

[0184] Embodiment 127 is an engineered cell as described in any one of Embodiments 103-126, wherein the gene modification in the TRAC contains at least one nucleotide within a genomic coordinate targeted by a TRAC guide RNA containing the guide sequence of SEQ ID NO: 413.

[0185] Embodiment 128 is an engineered cell as described in any one of Embodiments 103-127, wherein the gene modification comprises insertion / deletion, C-to-T substitution, or A-to-G substitution within the genomic coordinates.

[0186] Embodiment 129 is an engineered cell as described in any one of Embodiments 103-128, wherein the gene modification comprises C to T substitutions within the genomic coordinates.

[0187] Implementation scheme 130 is an engineered cell or cell population as described in any one of implementation schemes 42-127, 42.1, 42.2 and 44.1-44.4, wherein the cells are homozygous for HLA-C.

[0188] Implementation scheme 131 is an engineered cell or cell population as described in any one of implementation schemes 42-128, 42.1, 42.2 and 44.1-44.4, wherein the cells are homozygous for HLA-B and HLA-C.

[0189] Embodiment 132 is a cell population comprising engineered cells as described in any one of embodiments 103-131.

[0190] Embodiment 133 is a pharmaceutical composition comprising engineered cells or cell populations as described in any one of Embodiments 42-132, 42.1, 42.2 and 44.1-44.4.

[0191] Embodiment 134 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 42-133, 42.1, 42.2, and 44.1-44.4, wherein the genetic modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive nucleotides in the genomic coordinate system.

[0192] Embodiment 135 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 42-134, 42.1, 42.2, and 44.1-44.4, wherein the gene modification comprises an insertion / deletion.

[0193] Embodiment 136 is an engineered cell, cell population, or pharmaceutical composition as described in any one of embodiments 42-135, 42.1, 42.2, and 44.1-44.4, wherein the genetic modification comprises the insertion of a heterologous coding sequence.

[0194] Embodiment 137 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 42-136, 42.1, 42.2, and 44.1-44.4, wherein the genetic modification comprises at least one A to G substitution within the genomic coordinates.

[0195] Embodiment 138 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 42-137, 42.1, 42.2, and 44.1-44.4, wherein the genetic modification comprises at least one C to T substitution within the genomic coordinates.

[0196] Embodiment 139 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 42-138, 42.1, 42.2, and 44.1-44.4, wherein the cells have reduced expression of the TRAC protein on the surface of the cells.

[0197] Embodiment 140 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 42-139, 42.1, 42.2, and 44.1-44.4, wherein the cell has the genetic modification in the CIITA gene.

[0198] Embodiment 141 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 42-140, 42.1, 42.2, and 44.1-44.4, wherein the cells have reduced expression of MHC class II molecules on the surface of the cells.

[0199] Embodiment 142 is an engineered cell, cell population, or pharmaceutical composition as described in any one of embodiments 42-141, 42.1, 42.2, and 44.1-44.4, wherein the engineered cell is an immune cell.

[0200] Embodiment 143 is an engineered cell, cell population, or pharmaceutical composition as described in any one of embodiments 42-142, 42.1, 42.2, and 44.1-44.4, wherein the cell is an NK cell.

[0201] Embodiment 144 is an engineered cell, cell population, or pharmaceutical composition as described in any one of embodiments 42-142, 42.1, 42.2, and 44.1-44.4, wherein the cell is a T cell.

[0202] Embodiment 145 is an engineered cell, cell population, or pharmaceutical composition as described in Embodiment 144, wherein the T cell is a CD4+ T cell.

[0203] Embodiment 146 is an engineered cell, cell population, or pharmaceutical composition as described in Embodiment 144, wherein the T cell is a CD8+ T cell.

[0204] Embodiment 147 is an engineered cell, cell population, or pharmaceutical composition as described in Embodiment 144, wherein the T cells have a T memory stem cell (Tscm) phenotype.

[0205] Embodiment 148 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 42-141, 42.1, 42.2, and 44.1-44.4, wherein the engineered cell is a stem cell.

[0206] Embodiment 149 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 42-148, 42.1, 42.2, and 44.1-44.4, wherein the engineered cell is a primary cell.

[0207] Embodiment 150 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 42-149, 42.1, 42.2, and 44.1-44.4, wherein the cells are engineered using a genome editing system.

[0208] Embodiment 151 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 42-150, 42.1, 42.2, and 44.1-44.4, wherein the genome editing system comprises an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0209] Embodiment 152 is an engineered cell, cell population, or pharmaceutical composition as described in Embodiment 151, wherein the RNA-guided DNA binder or the RNA-guided DNA binder encoded by the nucleic acid is Streptococcus pyogenes (Streptococcus pyogenes). S. pyogenes Cas9 (SpyCas9).

[0210] Embodiment 153 is an engineered cell, cell population, or pharmaceutical composition as described in Embodiment 151, wherein the RNA-guided DNA binder or the RNA-guided DNA binder encoded by the nucleic acid is Neisseria meningitidis (Neisseria meningitidis). N. meningitidis Cas9 (NmeCas9).

[0211] Embodiment 154 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 151-153, wherein the RNA-guided DNA binder or the RNA-guided DNA binder encoded by the nucleic acid has double-stranded endonuclease activity.

[0212] Embodiment 155 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 151-153, wherein the RNA-guided DNA binder or the RNA-guided DNA binder encoded by the nucleic acid has nicking enzyme activity.

[0213] Embodiment 156 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 151-153, wherein the RNA-guided DNA binder or the RNA-guided DNA binder encoded by the nucleic acid comprises a dCas9 DNA-binding domain.

[0214] Embodiment 157 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 151-156, wherein the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is an A-to-G base editor.

[0215] Embodiment 158 ​​is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 151-156, wherein the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is a C-to-T base editor.

[0216] Embodiment 159 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 71-158, wherein the guide RNA is provided to the cell in a vector.

[0217] Embodiment 160 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 71-159, wherein the RNA-guided DNA binder is in a vector, optionally provided to the cell in the same vector as the guide RNA.

[0218] Embodiment 161 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 42-160, 42.1, 42.2, and 44.1-44.4, wherein the nucleic acid encoding the anti-CD70 CAR is provided to the cell in an expression vector.

[0219] Embodiment 162 is an engineered cell, cell population, or pharmaceutical composition as described in Embodiment 161, wherein the expression vector is a viral vector.

[0220] Embodiment 163 is an engineered cell, cell population, or pharmaceutical composition as described in Embodiment 162, wherein the expression vector comprises an AAV vector.

[0221] Embodiment 164 is an engineered cell, cell population, or pharmaceutical composition as described in Embodiment 163, wherein the expression vector comprises SEQ ID NO: 106.

[0222] Embodiment 165 is an engineered cell, cell population, or pharmaceutical composition as described in Embodiment 163, wherein the engineered cell comprises SEQ ID NO: 107.

[0223] Implementation scheme 166 is an engineered cell, cell population or pharmaceutical composition as described in implementation scheme 161, wherein the expression vector is a non-viral vector.

[0224] Embodiment 167 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 71-166, wherein the guide RNA is provided to the cell in a lipid nanoparticle (LNP), optionally in the same LNP that provides an RNA-guided DNA binder.

[0225] Embodiment 168 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 42-167, 42.1, 42.2, and 44.1-44.4, wherein the nucleic acid encoding the anti-CD70 CAR is provided to the cell in lipid nanoparticles (LNPs).

[0226] Embodiment 169 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 71-168, wherein the guide RNA is a single guide RNA.

[0227] Embodiment 170 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 71-169, wherein the guide RNA comprises a 5' end modification or a 3' end modification.

[0228] Embodiment 171 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 71-170, wherein the guide RNA comprises the sequence of SEQ ID NO: 712 or 722, or a sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 712 or 731.

[0229] Embodiment 172 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 71-170, wherein the guide RNA comprises the sequence of SEQ ID NO: 713 or 723, or a sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 713 or 723.

[0230] Embodiment 173 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 71-170, wherein the guide RNA comprises the sequence of SEQ ID NO: 620 or 658, or a sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 620 or 658.

[0231] Embodiment 174 is an engineered cell, cell population, or pharmaceutical composition as described in any one of Embodiments 71-170, wherein the guide RNA comprises a sequence of SEQ ID NO: 641 or 669, or a sequence having at least 90%, 95%, 98%, or 99% identity with SEQ ID NO: 641 or 669.

[0232] Implementation Scheme 175 is a method for manufacturing engineered cells, the method comprising contacting the cells with: a. a nucleic acid as described in any one of Implementation Schemes 25-32, or an mRNA as described in Implementation Scheme 33, or an expression vector as described in Implementation Scheme 34; and b. at least one genome editing tool comprising a genome editor and at least one guide RNA, wherein the at least one guide RNA targets a genomic locus selected from HLA-A, HLA-B, TRAC, CIITA, TGFBR2, and CD70 loci.

[0233] Embodiment 176 is a method for manufacturing engineered cells containing anti-CD70 CAR, the method comprising a. providing engineered cells having surface expression of one or both of TGFBR2 and CD70 reduced or eliminated relative to unmodified cells; and b. contacting the cells with a nucleic acid as described in any one of Embodiments 25-32, or an mRNA as described in Embodiment 33, or an expression vector as described in Embodiment 34.

[0234] Embodiment 177 is a method for manufacturing engineered cells containing anti-CD70 CAR, the method comprising a. providing engineered cells having surface expression of one or more of HLA-A, HLA-B, MHC class II, TRAC, TGFBR2 and CD70 reduced or eliminated relative to unmodified cells; and b. contacting the cells with a nucleic acid as described in any one of Embodiments 25-32, or an mRNA as described in Embodiment 33, or an expression vector as described in Embodiment 34.

[0235] Implementation scheme 178 is a method for manufacturing engineered cells containing anti-CD70 CAR, the method comprising: (a) contacting the cells with a first group of lipid nanoparticles (LNPs), the first group of LNPs comprising an LNP containing a UGI and at least one LNP containing a base editor and at least one guide RNA, the at least one guide RNA being homologous to the base editor and targeting a genomic locus selected from HLA-A, HLA-B, TRAC, CIITA, TGFBR2, and CD70 loci; and (b) contacting the cells with: i. a second group of LNPs, the second group of LNPs comprising an LNP containing a UGI and at least one LNP containing a base editor and at least one guide RNA. LNP, wherein the at least one guide RNA is homologous to the base editor and targets a genomic locus selected from HLA-A, HLA-B, TRAC, CIITA, TGFBR2, and CD70 loci, wherein the at least one guide RNA is different from the at least one guide RNA contained in the first set of LNPs of step (a); and ii. at least one LNP comprising an RNA-guided lyase and at least one gRNA, wherein the at least one gRNA is homologous to the RNA-guided lyase and targets the TRAC locus; and iii. nucleic acid encoding an anti-CD70 CAR for insertion into an editing site (e.g., a double-strand break) at the TRAC locus.

[0236] Implementation Scheme 179 is a method for manufacturing engineered cells containing anti-CD70 CAR, the method comprising: (a) contacting the cells with: a first group of lipid nanoparticles (LNPs), the first group of LNPs comprising a first LNP containing a base editor and gRNA targeting an HLA-A locus; a second LNP containing a base editor and gRNA targeting an HLA-B locus; a third LNP containing a base editor and gRNA targeting a CIITA locus; and a fourth LNP containing a uracil glycosidase inhibitor (UGI); (b) contacting the cells with: (i) the second group of LNPs, The second group of LNPs includes a fifth LNP containing a base editor and a gRNA targeting the TGFBR2 locus; a sixth LNP containing a base editor and a gRNA targeting the CD70 locus; (ii) a seventh LNP containing an RNA-guided DNA lyase and a gRNA homologous to the RNA-guided DNA lyase and targeting the TRAC locus; and an eighth lipid LNP containing a UGI; and (iii) a nucleic acid encoding anti-CD70CAR for insertion into an editing site (e.g., a double-strand break) at the TRAC locus.

[0237] Implementation scheme 180 is the method as described in implementation scheme 178 or 179, wherein the RNA-guided lyase comprises a Streptococcus pyogenes (Spy) Cas9 lyase, and the base editor comprises a Neisseria meningitidis (Nme) Cas9 nickase.

[0238] Implementation scheme 181 is a method of administering to a subject in need an engineered cell, cell population or pharmaceutical composition as described in any one of implementation schemes 42-174, 42.1, 42.2 and 44.1-44.4.

[0239] Implementation scheme 182 is a method of administering, as an adoptive cell transfer (ACT) therapy, to a subject an engineered cell, cell population, or pharmaceutical composition as described in any one of implementation schemes 42-174, 42.1, 42.2, and 44.1-44.4.

[0240] Implementation scheme 183 is a method for treating a disease or condition, the method comprising administering to a subject in need an engineered cell, cell population or pharmaceutical composition as described in any one of implementation schemes 42-174, 42.1, 42.2 and 44.1-44.4.

[0241] Implementation scheme 183.1 is the method as described in any one of implementation schemes 181 to 183, wherein the engineered cells are allogeneic to the subject.

[0242] Implementation scheme 184 is an engineered cell, cell population, composition or method as described in any one of implementation schemes 42-174, 42.1, 42.2 and 44.1-44.4, for administration to a subject as an adoptive cell transfer (ACT) therapy.

[0243] Implementation scheme 185 is an engineered cell, cell population, pharmaceutical composition or method as described in any one of implementation schemes 42-174, 42.1, 42.2 and 44.1-44.4, for the treatment of a subject suffering from cancer.

[0244] Implementation scheme 186 is an engineered cell, cell population or pharmaceutical composition as described in any one of implementation schemes 42-174, 42.1, 42.2 and 44.1-44.4, for the treatment of a subject suffering from an infectious disease.

[0245] Implementation scheme 187 is an engineered cell, cell population or pharmaceutical composition as described in any one of implementation schemes 42-174, 42.1, 42.2 and 44.1-44.4, for the treatment of a subject suffering from an autoimmune disease.

[0246] Implementation scheme 188 is the use of an engineered cell, cell population, or pharmaceutical composition as described in any one of implementation schemes 42-174, 42.1, 42.2, and 44.1-44.4, for the manufacture of a medicament for treating a subject suffering from cancer, an infectious disease, or an autoimmune disease.

[0247] Implementation scheme 189 is an engineered cell comprising gene modifications in the HLA-A gene, the HLA-B gene, the TRAC gene, the CIITA gene, the TGFBR2 gene, and / or the CD70 gene, wherein the gene modification in the HLA-A gene is located within genomic coordinates chr6:29942891-29942915; wherein the gene modification in the HLA-B gene is located within genomic coordinates chr6:31355222-31355246, chr6:31355221-31355245, or chr6:31355205-31355229; and wherein the gene modification in the TRAC gene is located within genomic coordinates chr14: Within 22547524-22547544; wherein the gene modification in the CIITA gene is within genomic coordinates chr16:10907504-10907528; wherein the gene modification in the TGFBR2 gene is within genomic coordinates chr3:30674205-30674229 or chr3:30671941-30671961; and wherein the gene modification in the CD70 gene is within genomic coordinates chr19:6590121-6590145 or chr19:6590998-6591018, wherein the engineered cells contain anti-CD70 chimeric antigen receptor (CAR) or contain nucleic acid encoding anti-CD70 CAR.

[0248] Implementation scheme 190 is an engineered cell comprising gene modifications in the HLA-A gene, the HLA-B gene, the TRAC gene, the CIITA gene, the TGFBR2 gene, and / or the CD70 gene, wherein the gene modifications in the HLA-A gene are located within genomic coordinates chr6:29942891-29942915; wherein the gene modifications in the HLA-B gene are located within genomic coordinates chr6:31355222-31355246; and wherein the gene modifications in the TRAC gene... The gene modification is located within genomic coordinates chr14:22547524-22547544; the gene modification in the CIITA gene is located within genomic coordinates chr16:10906643-10906667; the gene modification in the TGFBR2 gene is located within genomic coordinates chr3:30674205-30674229; and the gene modification in the CD70 gene is located within genomic coordinates chr19:6590121-6590145, wherein the engineered cell contains an anti-CD70 chimeric antigen receptor (CAR) or contains nucleic acid encoding an anti-CD70 CAR.

[0249] Embodiment 191 is an engineered cell as described in Embodiment 189 or 190, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 4, or wherein the nucleic acid encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 3 or 106.

[0250] Implementation Scheme 192 is an engineered human T cell comprising multiple genetic modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein (a) the engineered human T cell comprises genetic modifications to the HLA-A gene and reduced or eliminated HLA-A surface expression relative to unmodified cells, genetic modifications to the HLA-B gene and reduced or eliminated HLA-B surface expression relative to unmodified cells, genetic modifications to the CIITA gene and reduced or eliminated MHC class II surface expression relative to unmodified cells, genetic modifications to the TGFBR2 gene and reduced or eliminated TGFBR2 surface expression relative to unmodified cells, and genetic modifications to the CD70 gene and reduced or eliminated CD70 surface expression relative to unmodified cells, and (b) the anti-CD70 CAR comprises an antigen-binding protein or fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region, the heavy chain variable region comprising a complementarity-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, and comprising the amino acid sequence of SEQ ID NO: 73. The anti-CD70 CAR contains a VH CDR2 of amino acid sequence 74 and a VH CDR3 of amino acid sequence SEQ ID NO: 75, and contains a light chain variable (VL) region comprising a complementarity-determining region 1 (VL CDR1) containing amino acid sequence SEQ ID NO: 55, a VL CDR2 containing amino acid sequence SEQ ID NO: 56, and a VL CDR3 containing amino acid sequence SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted into the TRAC locus.

[0251] Implementation scheme 193 is an engineered human T cell comprising multiple gene modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein (a) the engineered human T cell comprises gene modifications within the genomic coordinates chr6:29942891-29942915 of the HLA-A gene, gene modifications within the genomic coordinates chr6:31355222-31355246 of the HLA-B gene, gene modifications within the genomic coordinates chr16:10906643-10906667 of the CIITA gene, gene modifications within the genomic coordinates chr3:30674205-30674229 of the TGFBR2 gene, and gene modifications within the genomic coordinates chr19:6590121-6590145 of the CD70 gene, and (b) the anti-CD70... The CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region comprising a complementation-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 75, and a light chain variable (VL) region comprising a complementation-determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 55, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VLCDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted into the TRAC locus.

[0252] Implementation scheme 194 is an engineered human T cell comprising multiple genetic modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein (a) the engineered human T cell comprises genetic modifications within the genomic coordinates chr6:29942891-29942915 of the HLA-A gene, genetic modifications within the genomic coordinates chr6:31355222-31355246 of the HLA-B gene, genetic modifications within the genomic coordinates chr16:10906643-10906667 of the CIITA gene, genetic modifications within the genomic coordinates chr3:30674205-30674229 of the TGFBR2 gene, and genetic modifications within the genomic coordinates chr19:6590121-6590145 of the CD70 gene, and (b) the anti-CD70... The CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region, the heavy chain variable region comprising a complementation-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 75, and a light chain variable (VL) region, the light chain variable region comprising a complementation-determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 55, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VLCDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted at genomic coordinates chr14:22547524-22547544 at the TRAC locus.

[0253] Embodiment 195 is an engineered human T cell as described in any one of Embodiments 192-194, wherein the VH region contains the amino acid sequence of SEQ ID NO: 45 and the VL region contains the amino acid sequence of SEQ ID NO: 39.

[0254] Embodiment 196 is an engineered human T cell as described in any one of Embodiments 192-195, wherein the anti-CD70 CAR comprises the amino acid sequence of SEQ ID NO: 4.

[0255] Implementation scheme 197 is an engineered human T cell as described in any one of implementation schemes 192-196, wherein the engineered human T cell is a CD4+ or CD8+ T cell.

[0256] Embodiment 198 is an engineered human T cell as described in any one of Embodiments 192-197, wherein the engineered human T cell is homozygous for HLA-C, and optionally wherein the engineered human T cell is homozygous for both HLA-B and HLA-C.

[0257] Implementation Scheme 199 is a pharmaceutical composition comprising a population of T cells, wherein the T cell population comprises CD4+ T cells and / or CD8+ T cells, the cells comprising engineered human T cells comprising multiple genetic modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein (a) the engineered human T cells comprise genetic modifications to the HLA-A gene and reduced or eliminated HLA-A surface expression relative to unmodified cells, genetic modifications to the HLA-B gene and reduced or eliminated HLA-B surface expression relative to unmodified cells, genetic modifications to the CIITA gene and reduced or eliminated MHC class II surface expression relative to unmodified cells, genetic modifications to the TGFBR2 gene and reduced or eliminated TGFBR2 surface expression relative to unmodified cells, and genetic modifications to the CD70 gene and reduced or eliminated CD70 surface expression relative to unmodified cells, and (b) the anti-CD70 CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region, the heavy chain variable region comprising SEQ ID NO: The anti-CD70 CAR contains a complementation-determining region 1 (VH CDR1) of the amino acid sequence of SEQ ID NO: 73, a VHCDR2 containing the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 75, and contains a light chain variable (VL) region comprising a complementation-determining region 1 (VL CDR1) containing the amino acid sequence of SEQ ID NO: 55, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 56, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted into the TRAC locus.

[0258] Implementation Scheme 200 is a pharmaceutical composition comprising a T cell population, wherein the T cell population comprises CD4+ T cells and / or CD8+ T cells, the cells comprising engineered human T cells containing multiple genetic modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein (a) the engineered human T cells comprise genetic modifications within genomic coordinates chr6:29942891-29942915 of the HLA-A gene, genetic modifications within genomic coordinates chr6:31355222-31355246 of the HLA-B gene, genetic modifications within genomic coordinates chr16:10906643-10906667 of the CIITA gene, genetic modifications within genomic coordinates chr3:30674205-30674229 of the TGFBR2 gene, and genetic modifications within genomic coordinates chr19:6590121-6590145 of the CD70 gene, and (b) the anti-CD70... The CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region, the heavy chain variable region comprising a complementarity-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 75, and a light chain variable (VL) region, the light chain variable region comprising a complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence of SEQ ID NO: 55, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted into the TRAC locus.

[0259] Implementation Scheme 201 is a pharmaceutical composition comprising a T cell population, wherein the T cell population comprises CD4+ T cells and / or CD8+ T cells, the cells comprising engineered human T cells containing multiple genetic modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein (a) the engineered human T cells comprise genetic modifications within genomic coordinates chr6:29942891-29942915 of the HLA-A gene, genetic modifications within genomic coordinates chr6:31355222-31355246 of the HLA-B gene, genetic modifications within genomic coordinates chr16:10906643-10906667 of the CIITA gene, genetic modifications within genomic coordinates chr3:30674205-30674229 of the TGFBR2 gene, and genetic modifications within genomic coordinates chr19:6590121-6590145 of the CD70 gene, and (b) the anti-CD70... The CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region, the heavy chain variable region comprising a complementarity-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 75, and a light chain variable (VL) region, the light chain variable region comprising a complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence of SEQ ID NO: 55, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted at genomic coordinates chr14:22547524-22547544 at the TRAC locus.

[0260] Embodiment 202 is a pharmaceutical composition as described in any one of Embodiments 199-201, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 45 and the VL region comprises the amino acid sequence of SEQ ID NO: 39.

[0261] Embodiment 203 is a pharmaceutical composition as described in any one of Embodiments 199-202, wherein the anti-CD70CAR comprises the amino acid sequence of SEQ ID NO: 4.

[0262] Embodiment 204 is a pharmaceutical composition as described in any one of embodiments 199-203, wherein the engineered human T cells are homozygous for HLA-C, and optionally wherein the engineered human T cells are homozygous for HLA-B and for HLA-C.

[0263] Implementation scheme 205 is a method of administering engineered human T cells or a pharmaceutical composition as described in any one of implementation schemes 192-204 to a subject in need or as an adoptive cell transfer (ACT) therapy.

[0264] Implementation scheme 206 is a method for treating a disease or condition, the method comprising administering to a subject in need an engineered human T cell or a pharmaceutical composition as described in any one of implementation schemes 192-204.

[0265] Embodiment 207 is an engineered human T cell or pharmaceutical composition as described in any one of Embodiments 192-204, for administration to a subject as an adoptive cell transfer (ACT) therapy, for treating a subject with cancer, for treating a subject with an infectious disease, or for treating a subject with an autoimmune disease.

[0266] Implementation scheme 208 is the method as described in implementation scheme 206, wherein the disease or condition is cancer.

[0267] Embodiment 209 is an engineered human T cell or pharmaceutical composition for use as described in Embodiment 207 or the method described in Embodiment 208, wherein the cancer is a solid tumor or a hematologic malignancy.

[0268] Implementation scheme 210 is an engineered human T cell or pharmaceutical composition or method for use as described in implementation scheme 209, wherein the solid tumor is renal cell carcinoma, or wherein the hematologic malignancy is acute myeloid leukemia or multiple myeloma.

[0269] I. Definition Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings: As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB if the order is important in the particular context. Continuing this example, it explicitly includes combinations containing repetitions of one or more items or items, such as BB, AAA, AAB, BBC, CBBA, CABA, etc. Those skilled in the art will understand that, unless the context otherwise makes it obvious, there is generally no limit to the number of items or items in any combination.

[0270] As used herein, the term “kit” refers to a package of related components, such as one or more polynucleotides or compositions, and one or more related materials, such as delivery devices (e.g., syringes), solvents, solutions, buffers, instructions, or desiccants.

[0271] As used herein, an "allogeneic" cell refers to a cell derived from a donor subject of the same species as the recipient subject, wherein the donor and recipient subjects are genetically different, for example, having different genes at one or more loci. Thus, for example, the cell is allogeneic to the subject to which the cell is to be administered. As used herein, cells removed or separated from a donor and not reintroduced into the original donor are considered allogeneic cells.

[0272] The term “allogeneic T-cell target” is used interchangeably herein and refers to proteins that mediate or contribute to host-to-graft or graft-to-host responses; and genes encoding said molecules and their associated regulatory elements (e.g., promoters). It will be understood that the term “allogeneic T-cell target” when used in conjunction with a target sequence or gRNA molecule refers to genes (and their associated regulatory elements) encoding allogeneic T-cell target proteins. Without being bound by theory, inhibiting or eliminating one or more allogeneic T-cell targets, for example, through the methods and compositions disclosed herein, can improve the efficacy, survival, function, and / or viability of allogeneic cells (e.g., allogeneic T cells), for example, by reducing or eliminating unwanted immunogenicity (such as host-to-graft or graft-to-host responses).

[0273] In a non-limiting example, the protein mediating or contributing to graft-versus-host response or host-versus-graft response is one or more components of a T-cell receptor. In one embodiment, a component of the T-cell receptor is a constant domain of T-cell receptor α, such as TCR α. In one embodiment, a component of the T-cell receptor is a T-cell receptor β chain, such as constant domain 1 or constant domain 2 of TCR β. Therefore, in embodiments where the protein encoded by an allogeneic T-cell target is a component of the TCR, the gene encoding the allogeneic T-cell target can be, for example, TRAC and combinations thereof.

[0274] In a non-limiting example, the protein that mediates or contributes to graft-versus-host response or host-versus-graft response is an HLA protein. Examples of HLA proteins include HLA-A and HLA-B. Therefore, in an embodiment where the allogeneic T cell target protein is an HLA protein, the gene encoding the allogeneic T cell target can be, for example, HLA-A, HLA-B, and combinations thereof.

[0275] In a non-limiting example, the protein mediating or contributing to graft-versus-host response or host-versus-graft response is a major histocompatibility complex class II (MHC II) molecule (e.g., HLA-Dx (where x refers to a letter of the MHC II protein, such as HLA-DM, HLA-DO, HLA-DR, HLA-DQ, and / or HLA-DP)), or a regulatory factor for the expression of MHC II, and combinations thereof. A non-limiting example is CIITA (also referred to herein as C2TA). Thus, in an embodiment where the allogeneic T cell target protein is CIITA, the gene encoding the allogeneic T cell target could be, for example, CIITA. As used herein, "autologous" cells refer to cells derived from the same subject, the material of which will be later reintroduced into said subject. Thus, for example, if cells are removed from a subject and then reintroduced into the same subject, said cells are considered autologous.

[0276] As used in the context of the CD70 protein, the term "CD70" refers to a cytokine belonging to the tumor necrosis factor (TNF) ligand family. As used in the context of nucleic acids, "CD70" refers to the gene encoding the CD70 protein molecule. The Human Genome Project has accession number NC_000019.10 (6581648..6591150).

[0277] As used herein, “CIITA” or “C2TA” refers to the nucleic acid or protein sequence of the “trans-activator of major histocompatibility complex class II”; the Human Genome Project has accession number NC_000016.10 (range 10866208..10941562), see GRCh38.p13. The CIITA protein in the cell nucleus acts as a positive regulator of MHC class II gene transcription and is essential for the expression of MHC class II proteins.

[0278] As used herein, “MHC” or “one or more MHC molecules” or “MHC protein” or “one or more MHC complexes” refers to a major histocompatibility complex molecule (or more), and includes, for example, MHC class I and MHC class II molecules. In humans, MHC molecules are referred to as “human leukocyte antigen” complexes or “HLA molecules” or “HLA proteins.” The use of the terms “MHC” and “HLA” is not intended to be restrictive; as used herein, the term “MHC” can be used to refer to human MHC molecules, i.e., HLA molecules. Therefore, the terms “MHC” and “HLA” are used interchangeably herein.

[0279] As used in the context of HLA-A proteins, the term "HLA-A" refers to an MHC class I protein molecule, which is a heterodimer (i.e., β-2 microglobulin) composed of a heavy chain (encoded by the HLA-A gene) and a light chain. As used in the context of nucleic acids, the term "HLA-A" or "HLA-A gene" refers to the gene encoding the heavy chain of the HLA-A protein molecule. The HLA-A gene is also known as "HLA class I histocompatibility, A α chain"; the Human Genome Project has accession number NC_000006.12 (29942532..29945870). The HLA-A gene is known to exist in thousands of different morphologies (also referred to as "alleles") in the population (and individuals can receive two different alleles of the HLA-A gene). Public databases of HLA-A alleles (including sequence information) can be found at IPD-IMGT / HLA: https: / / www.ebi.ac.uk / ipd / imgt / hla / Obtained. All HLA-A alleles are covered by the terms “HLA-A” and “HLA-A gene”.

[0280] As used in the context of nucleic acids in this article, "HLA-B" refers to the gene encoding the heavy chain of the HLA-B protein molecule. HLA-B is also known as "HLA class I histocompatibility, B α chain"; the Human Genome Project has accession number NC_000006.12 (31353875..31357179).

[0281] As used in the context of nucleic acids in this article, "HLA-C" refers to the gene encoding the heavy chain of the HLA-C protein molecule. HLA-C is also known as "HLA class I histocompatibility, Cα chain"; the Human Genome Project has accession number NC_000006.12 (31268749..31272092).

[0282] As used herein in the context of proteins, the term "TGFβR2" or "TGFBR2" refers to a transmembrane protein that has a protein kinase domain, forms a heterodimeric complex with transforming growth factor β (TGF-β) receptor type 1, and binds to TGF-β. As used herein in the context of nucleic acids, the term "TGFβR2" or "TGFBR2" refers to the gene encoding a protein molecule of transforming growth factor β (TGF-β) receptor type 2. The Human Genome Project has accession number NC_000003.12 (30606356..30694142).

[0283] As used in the context of TRAC proteins, the term "TRAC" refers to the T-cell receptor α-chain. As used in the context of nucleic acids, "TRAC" refers to the gene encoding the T-cell receptor α-chain. The human wild-type TRAC sequence is available at NCBI Gene ID: 28755; Ensembl: ENSG00000277734. T-cell receptor α constant, TCRA, IMD7, TRCA, and TRA are genetic synonyms for TRAC.

[0284] As used herein, the term "within genomic coordinates" includes the boundaries of a given range of genomic coordinates. For example, if chr6:29942854-chr6:29942913 is given, then the coordinates chr6:29942854-chr6:29942913 are covered. Throughout this application, the referenced genomic coordinates are based on genomic annotations from the human genome GRCh38 (also known as hg38) assembly from the Genome Reference Consortium, which are available on the website of the National Center for Biotechnology Information. Tools and methods for converting genomic coordinates between assemblies are known in the art and can be used to convert the genomic coordinates provided herein to corresponding coordinates in another assembly of the human genome, including conversion to earlier assemblies generated by the same institution or using the same algorithm (e.g., from GRCh38 to GRCh37), and conversion to assemblies generated by different institutions or algorithms (e.g., from GRCh38 to NCBI33, generated by the International Human Genome Sequencing Consortium). Available methods and tools known in the art include, but are not limited to, the NCBIG Genome Remapping Service, available at the National Center for Biotechnology Information; UCSC LiftOver, available at the UCSC Genome Brower; and Assembly Converter, available at Ensembl.org.

[0285] As used in this article, the term "homozygous" refers to two identical alleles that share a specific gene.

[0286] As used herein, the term “subject” is intended to include living organisms in which an immune response may be triggered, including, for example, mammals, primates, and humans.

[0287] "Polynucleotide" and "nucleic acid" are used herein to refer to polymeric compounds containing nucleosides or nucleoside analogs having nitrogen-containing heterocyclic bases or base analogs linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers as analogs thereof. The nucleic acid "backbone" can consist of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide-nucleic acid" or PNA; PCT No. WO 95 / 32305), thiophosphate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of the nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions (e.g., 2'-methoxy or 2'-halide substitutions). The nitrogenous bases can be conventional bases (A, G, C, T, U), their analogs (e.g., modified uridines, such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine, or others); inosine; purine or pyrimidine derivatives (e.g., N... 4 -Methyldeoxyguanosine, denitro- or aza-purine, denitro- or aza-pyrimidine, pyrimidine bases with substituents at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with substituents at the 2, 6, or 8 position, 2-amino-6-methylaminopurine, O 6 -Methylguanine, 4-thiopyrimidine, 4-aminopyrimidine, 4-dimethylhydrazine-pyrimidine and O 4 -alkyl-pyrimidine; US Patent No. 5,378,825 and PCT No. WO 93 / 13121). For general discussion, see [link to general discussion]. The Biochemistry of the Nucleic Acids 5-36, Adams et al., eds., 11th ed., 1992. Nucleic acids may include one or more “base-free” residues, wherein the backbone does not include nitrogenous bases at one or more positions of the polymer (US Patent No. 5,585,481). Nucleic acids may contain only conventional RNA or DNA sugars, bases, and linkages, or may include conventional components and substitutions for both (e.g., conventional bases with 2'-methoxy linkages, or polymers containing conventional bases and one or more base analogs). Nucleic acids include “locked nucleic acids” (LNAs), which are analogs containing one or more LNA nucleotide monomers, wherein the bicyclic furanose unit is locked in a glycosidic conformation mimicking RNA, which enhances hybridization affinity for complementary RNA and DNA sequences (Vester and Wengel, 2004). Biochemistry 43(42):13233-41). RNA and DNA have different sugar moieties and may differ due to the presence of uracil or its analogues in RNA and thymine or its analogues in DNA.

[0288] The terms “guide RNA,” “gRNA,” and simply “guide” are used interchangeably in this document to refer to, for example, a guide RNA that directs a DNA-binding agent to a target DNA, and can be a single guide RNA or a combination of crRNA and trRNA (also known as tracrRNA). Exemplary gRNAs include class II Cas nuclease guide RNAs in modified or unmodified forms. crRNA and trRNA can associate to form a single RNA molecule (single guide RNA, sgRNA) or two separate RNA strands (dual guide RNA, dgRNA). “Guide RNA” or “gRNA” refers to each type. trRNA can be a naturally occurring sequence or a modified or altered trRNA sequence compared to a naturally occurring sequence.

[0289] As used herein, a "guide sequence" refers to a sequence within the guide RNA that is complementary to the target sequence and functions as an RNA-guided DNA binder to direct the guide RNA toward the target sequence for binding or modification (e.g., cleavage). The "guide sequence" may also be referred to as a "target sequence" or a "spacer sequence." Guide sequences can be 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, for example, in *Neisseria meningitidis*. (Neisseria meningitides)In some embodiments, the Nme Cas9 guide sequence comprises at least 22, 23, or 24 consecutive nucleotides selected from SEQ ID NO: 2-80, 101-120, 201, 265, 301, 302, 304-576, and 601-774. In some embodiments, the target sequence is, for example, in a gene or on a chromosome, and is complementary to the guide sequence. In some embodiments, the complementarity or identity between the guide sequence and its corresponding target sequence is at least 80%, 85%, 90%, or 95%. For example, in some embodiments, the guide sequence comprises 24 consecutive nucleotides selected from SEQ ID NO: 2-80, 101-120, 201, 265, 301, 302, 304-576, and 601-774. In some embodiments, the guide sequence may have 100% complementarity or identity with the target region. In other embodiments, the guide sequence and target region may contain at least one mismatch, i.e., a non-identical or non-complementary nucleotide, depending on the reference sequence. For example, the guide sequence and target sequence may contain 1-2, preferably no more than 1, mismatches, wherein the total length of the target sequence is 19, 20, 21, 22, 23, or 24 or more nucleotides. In some embodiments, the guide sequence and target region may contain 1-2 mismatches, wherein the guide sequence contains at least 24 or more nucleotides. In some embodiments, the guide sequence and target region may contain 1-2 mismatches, wherein the guide sequence contains 24 nucleotides. That is, the guide sequence and target region may form a double-stranded region with one or more base pairs. In some embodiments, the double-stranded region may include 1-2 mismatches such that the guide strand is not perfectly complementary to the target sequence. Mismatch locations are known in the art; for example, distal mismatches in PAM are often more tolerable than proximal matches in PAM. Mismatch tolerance at other locations is known in the art (see, for example, Edraki et al., 2019. Mol. Cell, 73:1-13).

[0290] The target sequences of RNA-guided DNA binders include both the positive and negative strands of genomic DNA (i.e., a given sequence and its inverse complement), because the nucleic acid substrates of RNA-guided DNA binders are double-stranded nucleic acids. Therefore, in cases where the guide sequence is described as "complementary to the target sequence," it should be understood that the guide sequence can direct the guide RNA to bind to the inverse complement of the target sequence. Thus, in some embodiments, in the case of the guide sequence binding to the inverse complement of the target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., a target sequence excluding PAM), except that T is replaced with U in the guide sequence.

[0291] As used herein, “RNA-guided DNA binder” means a polypeptide or polypeptide complex with RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the presence of a PAM and the sequence of the guide RNA. Exemplary RNA-guided DNA binders include Cas lysins / nicking enzymes and their inactive forms (“dCas DNA binders”). As used herein, “Cas nuclease” is also referred to as “Cas protein” and encompasses Cas lysins, Cas nicking enzymes, and dCas DNA binders. Cas lysins / nicking enzymes and dCas DNA binders include the Csm or Cmr complex of the type III CRISPR system, its Cas10, Csm1, or Cmr2 subunits, the cascade complex of the type I CRISPR system, its Cas3 subunit, and class II Cas nucleases.

[0292] As used herein, “class 2 Cas nucleases” are single-stranded polypeptides with RNA-guided DNA-binding activity. Class 2 Cas nucleases include class 2 Cas lyases / nickases (e.g., H840A, D10A, or N863A variants of SpyCas9, and NmeCas9, such as D16A and H588A of Nme2 Cas9), which further have RNA-guided DNA lyase or nickase activity, and class 2 dCas DNA binders, wherein the lyase / nickase activity is inactive. Class 2 Cas nucleases include proteins such as Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) and their modifications. Cpf1 protein (Zetsche et al., Cell (163: 1-13 (2015)) is homologous to Cas9 and contains a RuvC-like nuclease domain. Zetsche's Cpf1 sequence is incorporated in its entirety by reference. See, for example, Zetsche, Tables S1 and S3. See, for example, Makarova et al. Nat Rev Microbiol , 13(11): 722-36 (2015); Shmakov et al., Molecular Cell , 60:385-397 (2015).

[0293] Several Cas9 heterologs have been obtained from Neisseria meningitidis (Esvelt et al., NAT. METHODS, Vol. 10, 2013, 1116-1121; Hou et al., PNAS, Vol. 110, 2013, pp. 15644-15649) (Nme1Cas9, Nme2Cas9, and Nme3Cas9). The Nme2Cas9 heterolog functions effectively in mammalian cells, recognizes N4CC PAM, and can be used for in vivo editing using homologous gRNAs (Ran et al., NATURE, Vol. 520, 2015, pp. 186-191; Kim et al., NAT. COMMUN., Vol. 8, 2017, p. 14500). Nme2Cas9 can be specific and selective, for example, capable of low-off-target editing (Lee et al., MOL. THER., Vol. 24, 2016, pp. 645–654; Kim et al., 2017). See also WO / 2020081568 (e.g., pp. 28 and 42), which describes the Nme2Cas9 D16A nickase, the contents of which are hereby incorporated in their entirety by reference. Throughout this text, “NmeCas9” or “NmeCas9” is generic and encompasses any type of NmeCas9, including Nme1Cas9, Nme2Cas9, and Nme3Cas9.

[0294] Table 10 provides exemplary nucleotide and polypeptide sequences of the Cas9 molecule. Methods for identifying alternative nucleotide sequences (including naturally occurring alternative variants) encoding the Cas9 polypeptide sequence are known in the art. Also covered are sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with either the Cas9 nucleic acid sequence or any nucleic acid sequence encoding the amino acid sequence provided herein.

[0295] As used herein, the term "editor" refers to an agent containing a polypeptide capable of modifying a DNA sequence. In some embodiments, the editor is a lysin, such as Cas9 lysin. In some embodiments, the editor is capable of deaminating bases within a DNA molecule, and it may be referred to as a base editor. In some embodiments, the editor is capable of deaminating cytosine (C) in DNA. In some embodiments, the editor is a fusion protein containing an RNA-guided nicking enzyme fused with cytidine deaminase. In some embodiments, the editor is a fusion protein containing an RNA-guided nicking enzyme fused with APOBEC3A deaminase (A3A). In some embodiments, the editor contains a Cas9 nicking enzyme fused with APOBEC3A deaminase (A3A). In some embodiments, the editor is a fusion protein containing an RNA-guided nicking enzyme fused with cytidine deaminase and UGI. In some embodiments, the editor lacks UGI.

[0296] As used herein, “cytidine deaminase” refers to a polypeptide or polypeptide complex that possesses cytidine deaminase activity, catalyzing the hydrolytic deamination of cytidine or deoxycytidine, typically producing uridine or deoxyuridine. Cytidine deaminases encompass enzymes in the cytidine deaminase superfamily, and particularly enzymes of the APOBEC family (enzymes of the APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups), activation-induced cytidine deaminases (AID or AICDA), and CMP deaminases (see, for example, Conticello et al., Mol. Biol. Evol. 22:367-77, 2005; Conticello, Genome Biol. 9:229, 2008; Muramatsu et al., J. Biol. Chem. 274: 18470-6, 1999; Carrington et al., Cells 9:1690(2020)). In some embodiments, variants of any known cytidine deaminase or APOBEC protein are included. Variants include proteins having sequences that differ from wild-type proteins due to one or more mutations (i.e., substitution, deletion, insertion), such as one or more single-point substitutions. For example, shortened sequences can be used, for instance, by deleting the N-terminus, C-terminus, or internal amino acids, preferably deleting one to four amino acids from the C-terminus. As used herein, the term "variant" refers to allelic variants, splicing variants, and natural or artificial mutants that are homologous to a reference sequence. Variants are "functional" because they exhibit catalytic activity for DNA editing.

[0297] As used herein, the term "APOBEC3A" refers to a cytidine deaminase, such as a protein expressed by the human A3A gene. APOBEC3A can possess catalytic DNA editing activity. The amino acid sequence of APOBEC3A has been described (UniPROT accession ID: p31941) and is included herein as SEQ ID NO: 850. In some embodiments, the APOBEC3A protein is the human APOBEC3A protein or the wild-type protein. Variants include proteins having a sequence that differs from the wild-type APOBEC3A protein due to one or more mutations (i.e., substitution, deletion, insertion), such as one or more single-point substitutions. For example, a shortened APOBEC3A sequence can be used, for instance, by deleting the N-terminal, C-terminal, or internal amino acids, preferably deleting one to four amino acids from the C-terminus of the sequence. As used herein, the term "variant" refers to an allelic variant, splice variant, and natural or artificial mutant homologous to the APOBEC3A reference sequence. A variant is "functional" because it exhibits catalytic activity for DNA editing. In some embodiments, APOBEC3A (such as human APOBEC3A) has wild-type amino acid position 57 (as numbered in the wild-type sequence). In some embodiments, APOBEC3A (such as human APOBEC3A) has asparagine at amino acid position 57 (as numbered in the wild-type sequence).

[0298] As used herein, a “nicking enzyme” is an enzyme that produces single-strand breaks (also known as “nicks”) in double-stranded DNA (i.e., cuts one strand of the DNA double helix but not the other). As used herein, “RNA-guided DNA nicking enzyme” refers to a polypeptide or polypeptide complex with DNA nicking enzyme activity, wherein the DNA nicking enzyme activity is sequence-specific and depends on the RNA sequence. Exemplary RNA-guided DNA nicking enzymes include Cas nicking enzymes. Class 2 Cas nicking enzymes include polypeptides with inactive HNH or RuvC catalytic domains, such as Cas9 (e.g., variants of SpyCas9 H840A, D10A, or N863A, or variant of NmeCas9 D16A). Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain or RuvC or RuvC-like domain of Neisseria meningitidis include Nme2Cas9 D16A (HNH nicking enzyme) and Nme2Cas9 H588A (RuvC nicking enzyme). Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and their modifications. Cpf1 protein (Zetsche et al., Cell The Cpf1 sequence (163: 1-13 (2015)) is homologous to Cas9 and contains a RuvC-like protein domain. Zetsche's Cpf1 sequence is incorporated in its entirety by reference. See, for example, Zetsche, Tables S1 and S3. "Cas9" encompasses *Spy* Cas9, the Cas9 variants listed herein, and their equivalents. See, for example, Makarova et al., *Nat RevMicrobiol*, 13(11): 722-36 (2015); Shmakov et al., *Molecular Cell*, 60:385-397 (2015).

[0299] As used herein, the term "fusion protein" refers to a hybrid polypeptide containing protein domains derived from at least two different proteins. One protein may be located at the N-terminal (N-terminal) portion or the C-terminal (C-terminal) portion of the fusion protein, thereby forming an "N-terminal fusion protein" or a "C-terminal fusion protein," respectively. Any protein presented herein can be produced by any method known in the art. For example, the proteins presented herein can be produced via recombinant protein expression and purification, particularly suitable for fusion proteins containing peptide linkers. Methods for recombinant protein expression and purification are well known and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), the entire contents of which are incorporated herein by reference.

[0300] The term "chimeric antigen receptor" or alternatively "CAR" refers to a group of polypeptides, typically two polypeptides in the simplest embodiment, that provide specificity to target cells, typically cancer cells, and provide intracellular signaling when present in immune effector cells. In some embodiments, the CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain (also referred to herein as "intracellular domain"), said intracellular domain comprising functional signaling domains derived from stimulatory molecules (e.g., activation domains) and / or costimulatory molecules (e.g., costimulatory domains) as defined below. In some aspects, the polypeptides are adjacent to each other. In some embodiments, the polypeptides include a dimerization switch that can couple the polypeptides to each other in the presence of a dimerizing molecule, for example, coupling the antigen-binding domain to the intracellular domain. In one aspect, the stimulatory molecule is a ζ-chain associated with a T-cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one aspect, the co-stimulatory molecule is selected from the co-stimulatory molecules described herein, such as 41BB (i.e., CD137), CD27, and / or CD28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, the intracellular domain comprising a functional signaling domain derived from the stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, the intracellular domain comprising a functional signaling domain derived from the co-stimulatory molecule and a functional signaling domain derived from the stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, the intracellular domain comprising two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from the stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, the intracellular domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR includes an optional leader sequence at its N-terminus (N-terminus). In another aspect, the CAR further includes a leader sequence at the N-terminus of the extracellular antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen-binding domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

[0301] CARs containing an antigen-binding domain (e.g., scFv or TCR) that targets a specific tumor marker X, such as those described herein, are also referred to as anti-X CARs. For example, a CAR containing an antigen-binding domain that targets CD19 is called an anti-CD19 CAR. As another example, a CAR containing an antigen-binding domain that targets BCMA is called an anti-BCMACAR. The term "signal transduction domain" refers to the functional part of a protein that functions by transmitting information within the cell to regulate cellular activity via defined signal transduction pathways by generating a second messenger or to act as an effector by responding to such messengers.

[0302] As used herein, the term "linker" refers to a chemical group or molecule that connects two adjacent molecules or portions. Typically, a linker is located between or on either side of two groups, molecules, or other portions and is covalently linked to each other. In some embodiments, the linker is one or more amino acids (e.g., a peptide or protein), such as the 16-amino acid residue "XTEN" linker or a variant thereof (see, for example, examples; and Schellenberger et al., A recombinant polypeptide extends the in vivo half-life of peptides and proteins in atunable manner. Nat. Biotechnol. 27, 1186-1190 (2009)). In some embodiments, the XTEN linker comprises the sequence SGSETPGTSESATPES (SEQ ID NO: 901), SGSETPGTSESA (SEQ ID NO: 902), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 903). In some embodiments, the linker is a peptide linker comprising one or more sequences selected from SEQ ID NO: 901-991.

[0303] As used herein, the term "uracil glycosidase inhibitor" or "UGI" refers to a protein that can inhibit the uracil-DNA glycosidase (UDG) base excision repair enzyme.

[0304] As used herein, an "open reading frame" or "ORF" of a gene refers to a sequence of codons that specify the amino acid sequence of the protein encoded by the gene. An ORF begins with a start codon (e.g., ATG in DNA or AUG in RNA) and ends with a stop codon (e.g., TAA, TAG, or TGA in DNA, or UAA, UAG, or UGA in RNA).

[0305] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA along with an RNA-guided DNA binder, such as a Cas nuclease, for example a Cas lyase, a Cas nickase, or a dCas DNA binder (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA binder (such as Cas9) to a target sequence, and the guide RNA hybridizes to the target sequence and the binder binds to the target sequence; where the binder is a lyase or a nickase, cleavage or nicking can occur after binding.

[0306] As used herein, a first sequence is considered to "contain a sequence with at least X% identity to the second sequence" if an alignment of the first and second sequences shows that X% or more positions in the entire second sequence match the first sequence. For example, the sequence AAGA contains a sequence with 100% identity to the sequence AAG because the alignment would result in 100% identity due to matching all three positions in the second sequence. Differences between RNA and DNA (generally uridine exchanged for thymidine and vice versa) and the presence of nucleoside analogs (such as modified uridine) do not result in differences in identity or complementarity between polynucleotides, provided that the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., for all of thymidine, uridine, or modified uridine, it is adenosine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as complement). Therefore, for example, the sequence 5'-AXG (where X is any modified uridine, such as pseudouridine, N1-methylpseudouridine, or 5-methoxyuridine) is considered to have 100% identity with AUG because both are perfectly complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. Those skilled in the art will understand which algorithm and parameter settings are appropriate for a given sequence pair to be aligned; for sequences generally of similar length and expected identity (>50% for amino acids or >75% for nucleotides), the Needleman-Wunsch algorithm with its preset settings provided by EBI on the www.ebi.ac.uk web server is generally suitable.

[0307] "Messenger RNA" or "mRNA" as used herein refers to a polynucleotide that contains an open reading frame that can be translated into a polypeptide (i.e., can be used as a translation substrate for ribosomes and aminoacylated tRNA). mRNA may contain one or more modifications, as provided below.

[0308] As used herein, “genetic modification” is a change at the DNA level, induced, for example by CRISPR / Cas9 gRNA and the Cas9 system. Genetic modifications can involve insertions, deletions, or substitutions (i.e., base sequence substitutions, i.e., mutations), typically within a defined sequence or genomic locus. Genetic modifications alter the nucleic acid sequence of DNA. Genetic modifications can occur at a single nucleotide position. Genetic modifications can occur at multiple nucleotide positions, such as 2, 3, 4, 5, or more nucleotides, typically adjacent to each other, such as consecutive nucleotides. Genetic modifications can occur in coding sequences, such as exon sequences. Genetic modifications can occur at splice sites, i.e., sufficiently close to a splice acceptor or splice donor site to disrupt splicing. Genetic modifications can include insertions of nucleotide sequences that are not endogenous to a genomic locus, such as insertions of heterologous open reading frames or coding sequences of genes. As used herein, genetic modifications can be used to prevent the translation of endogenous full-length proteins that have the amino acid sequence of a full-length protein prior to genetic modification of a genomic locus. Preventing the translation of endogenous full-length proteins or gene products includes preventing the translation of proteins or gene products of any length. Translation of endogenous full-length proteins can be prevented, for example, by frameshift mutations that lead to premature stop codon generation or by the generation of meaningless mutations. Translation of endogenous full-length proteins can be prevented by disrupting splicing. Translation of endogenous full-length proteins can be prevented by inserting heterologous coding sequences. Translation of endogenous full-length proteins can be prevented by altering the coding sequence at one or more locations to provide a modified full-length coding sequence different from the endogenous sequence present in the cell (e.g., correction of point mutations) (e.g., when the endogenous full-length protein contains unwanted mutations). Translation of endogenous full-length proteins can be prevented by altering the splicing of the endogenous full-length protein to produce different proteins through alternative splicing.

[0309] As used herein, “insertion / deletion” refers to an insertion or deletion mutation consisting of multiple nucleotides that are inserted, deleted, or inserted and deleted in a target nucleic acid (e.g., at a double-strand break (DSB) site). As used herein, when an insertion / deletion results in an insertion, the insertion is a random insertion at a DSB site and may or may not be guided by or based on a template sequence.

[0310] As used herein, a “heterologous coding sequence” refers to a coding sequence introduced into a cell as a foreign source (e.g., inserted into a genomic locus, such as a safe harbor locus, including the TCR locus). That is, the introduced coding sequence is heterologous, at least with respect to its insertion site. A polypeptide expressed by a gene from such a heterologous coding sequence is called a “heterologous polypeptide.” Heterologous coding sequences can be naturally occurring or engineered, and can be wild-type or variants. Heterologous coding sequences can include nucleotide sequences different from those encoding the heterologous polypeptide (e.g., internal ribosome entry sites). Heterologous coding sequences can be coding sequences that are naturally occurring in the genome as wild-type or variants (e.g., mutants). For example, although a cell contains the coding sequence of interest (as wild-type or as a variant), the same coding sequence or a variant thereof can be introduced as a foreign source, for example, expressed at a highly expressed locus. Heterologous coding sequences can also be coding sequences that are not naturally occurring in the genome, or coding sequences that express heterologous polypeptides that are not naturally occurring in the genome. “Heterologous coding sequence,” “foreign coding sequence,” and “transgenic” are used interchangeably. In some embodiments, the heterologous coding sequence or transgene includes a foreign nucleic acid sequence, such as a nucleic acid sequence that is not endogenous to the recipient cell. For example, the heterologous coding sequence may be heterologous relative to its insertion site and relative to its recipient cell.

[0311] As used herein, “reduced or eliminated” expression of a protein on a cell refers to a partial or complete loss of protein expression relative to unmodified cells. In some embodiments, the surface expression of a protein on a cell is measured by flow cytometry, and “reduced” or “eliminated” surface expression relative to unmodified cells is demonstrated by a reduction in fluorescence signal after staining with the same antibody targeting the protein. Cells found by flow cytometry to have “reduced” or “eliminated” protein surface expression relative to unmodified cells can be described as having “negative” expression for that protein, as demonstrated by a fluorescence signal similar to that of cells stained with an isotype control antibody. The “reduction” or “elimination” of protein expression can be measured using other techniques known in the art, utilizing appropriate controls known to those skilled in the art.

[0312] As used herein, “knockdown” refers to, for example, a reduction in the expression of a specific gene target or a protein (e.g., protein, mRNA, or both) encoded by the gene target compared to the expression of an unedited target sequence. Protein knockdown can be measured by detecting the total cellular amount of protein from a sample, such as a tissue, fluid, or cell population of interest. Protein knockdown can also be measured by measuring protein substitutes, markers, or activity. Methods for measuring mRNA knockdown are known and involve analyzing mRNA isolated from the sample of interest. In some embodiments, “knockdown” can refer to some loss of expression of a specific gene target, such as a reduction in the amount of transcribed mRNA or a reduction in the amount of protein expressed by cells or cell populations, including in vivo populations such as those found in tissues.

[0313] As used in this article, "knockout" refers to the loss of expression of a specific gene or protein in a cell. Knockout can cause expression to drop below the detectable level. Knockout can be measured by detecting the total cellular amount of the protein in a cell, tissue, or cell population.

[0314] As used herein, "target sequence" or "genomic target sequence" refers to a nucleic acid sequence in a target gene that is complementary to the guide sequence of the gRNA. The interaction between the target sequence and the guide sequence directs the binding of an RNA-guided DNA binder within the target sequence and potentially causes cleavage or splitting (depending on the activity of the binder).

[0315] As used herein, “treatment” means any administration or application of a therapeutic agent to a subject’s disease or condition, and includes suppressing the disease, halting the progression of the disease, alleviating one or more symptoms of the disease, curing the disease, or preventing one or more symptoms of the disease (including recurrence of symptoms).

[0316] Reference will now be made in detail to certain embodiments of this disclosure, examples of which are illustrated in the accompanying drawings. Although this disclosure is described in conjunction with the illustrated embodiments, it will be understood that the embodiments described are not intended to limit this disclosure to those embodiments. Rather, this disclosure is intended to cover all alternatives, modifications, and equivalents that may be included within this disclosure as defined in the appended claims and the included embodiments.

[0317] Before describing the teachings of this invention in detail, it should be understood that this disclosure is not limited to specific compositions or process steps and is therefore subject to variation. It should be noted that, unless the context clearly indicates otherwise, the singular forms “a / an” and “described” as used in this specification and the appended claims include multiple references. Thus, for example, a reference to “conjugate” includes multiple conjugates and a reference to “cell” includes multiple cells, etc.

[0318] Numerical ranges include the numbers that define the range. Taking into account significant digits and measurement-related errors, measured and measurable values ​​should be understood as approximate. Furthermore, the use of "comprise," "contain," and "include" is not intended to be restrictive. It should be understood that the foregoing general and detailed descriptions are illustrative and explanatory only, and not intended to limit the teaching.

[0319] Unless otherwise specified in this specification, embodiments described as "comprising" various components are also considered to be "consisting of the described components" or "substantially consisting of the described components"; embodiments described as "consisting of various components" are also considered to "comprising" the described components or "substantially consisting of the described components"; and embodiments described as "substantially consisting of various components" are also considered to "consisting of" or "comprising" the described components (this interchangeability does not apply to the use of these terms in the claims). Unless the context clearly indicates otherwise, the term "or" is used in an inclusive sense, i.e., equivalent to "and / or".

[0320] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter in any way. In the event of any material incorporated by reference that contradicts any terminology defined herein or any other expression herein, this specification shall prevail. Although the teachings of this invention have been described in conjunction with various embodiments, it is not intended to limit the teachings to such embodiments. Rather, as those skilled in the art will understand, the teachings of this invention encompass various alternatives, modifications, and equivalents.

[0321] II. Anti-CD70 chimeric antigen receptor (CAR) This disclosure describes a novel CAR comprising an anti-CD70 antibody fused to a CAR scaffold and generally having different transmembrane and intracellular domains. The resulting CAR provides enhanced cytotoxicity against tumor cells both in vitro and in vivo.

[0322] Generally, aspects of this disclosure relate to or include isolated nucleic acid molecules encoding anti-CD70 CARs, wherein the anti-CD70 CAR comprises an antigen-binding domain (e.g., an antibody or antibody fragment), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular domain (e.g., an intracellular domain described herein) that binds to a tumor antigen (i.e., CD70) as described herein (e.g., an intracellular domain comprising a co-stimulatory domain (e.g., a co-stimulatory domain described herein) and / or a primary signaling domain (e.g., an activation domain described herein)). In other aspects, this disclosure includes host cells containing the aforementioned nucleic acids and isolated proteins encoded by such nucleic acid molecules. CAR-containing nucleic acid constructs, vectors encoding the proteins, host cells, pharmaceutical compositions, and methods of administration and treatment related to this disclosure are disclosed in detail in International Patent Application Publication No. WO2015142675, which is incorporated herein by reference in its entirety.

[0323] In one aspect, this disclosure relates to isolated nucleic acid molecules encoding anti-CD70 CARs, wherein the anti-CD70 CAR comprises an antigen-binding domain (e.g., an antibody or antibody fragment), a transmembrane domain (e.g., a transmembrane domain as described herein), and an intracellular domain (e.g., an intracellular domain as described herein) that binds to a tumor-supporting antigen (e.g., a tumor-supporting antigen, such as CD70 as described herein) (e.g., an intracellular domain comprising a co-stimulatory domain (e.g., a co-stimulatory domain as described herein) and / or a primary signaling domain (e.g., an activation domain as described herein). In other aspects, this disclosure relates to polypeptides encoded by such nucleic acids and host cells containing such nucleic acids and / or polypeptides.

[0324] This disclosure provides cells, such as immune effector cells (e.g., T cells, NK cells), containing gRNA molecules or CRISPR systems as described herein, or containing genetic modifications within genomic coordinates targeted by guide RNAs or CRISPR systems as described herein, to be further engineered to contain one or more anti-CD70 CARs that direct immune effector cells to unwanted cells (e.g., cancer cells). This is achieved via an antigen-binding domain on the anti-CD70 CAR that is specific to cancer-associated antigens (e.g., CD70).

[0325] The disclosed anti-CD70 CAR also includes a transmembrane domain or a functional fragment thereof. In some embodiments, the transmembrane domain is a CD8a or CD28 transmembrane domain. The disclosed anti-CD70 CAR also includes a hinge domain or a functional fragment thereof between the antigen-binding protein and the transmembrane domain.

[0326] The disclosed anti-CD70 CAR may further comprise an intracellular domain comprising one or more of an activation domain and / or a co-stimulatory signaling domain (or co-stimulatory domain). In some embodiments, the intracellular domain comprises a sequence encoding an activation domain. In some embodiments, the intracellular domain comprises a co-stimulatory signaling domain. In some embodiments, the intracellular domain comprises both an activation domain and a co-stimulatory signaling domain. In some embodiments, the intracellular domain comprises a co-stimulatory domain or a functional fragment thereof, wherein the co-stimulatory domain is a 4-1BB or CD28 co-stimulatory domain.

[0327] This disclosure also covers isolated nucleic acid molecules comprising sequences encoding the disclosed amino acid sequences and CARs. It should be noted that, where an amino acid sequence is described, the nucleic acid sequence encoding said amino acid sequence is also included.

[0328] The disclosed anti-CD70 CAR also includes a transmembrane domain or a functional fragment thereof. In some embodiments, the transmembrane domain is a CD8a or CD28 transmembrane domain.

[0329] In some embodiments, this disclosure provides engineered cells comprising nucleic acids, mRNA, or expression vectors. In some embodiments, this disclosure provides engineered cells comprising anti-CD70 CAR. In some embodiments, this disclosure provides engineered cells comprising anti-CD70 CAR, wherein the cells are transduced with an expression vector operatively linked to or comprising a nucleic acid encoding the anti-CD70 CAR, and wherein the expression vector directs the expression of anti-CD70 CAR in the cells. In some embodiments, this disclosure provides expression vectors or engineered cells, wherein the expression vector comprises a retroviral or lentiviral expression vector.

[0330] Exemplary anti-CD70 CAR peptide and nucleic acid sequences are shown in Tables 1A and 1B below.

[0331] Table 1A. Exemplary anti-CD70 CARs

[0332] Table 1B. Exemplary anti-CD70 CARs

[0333] In some embodiments, this disclosure provides an anti-CD70 chimeric antigen receptor (CAR) comprising: (a) an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises: a heavy chain variable (VH) region comprising complementarity-determining region 1 (VH CDR1), VH CDR2, and VH CDR3, and a light chain variable (VL) region comprising complementarity-determining region 1 (VL CDR1), VL CDR2, and VL CDR3, and wherein: (i) the VH CDR1 comprises the amino acid sequence of any one of SEQ ID NO: 67, 70, 73, 76, 79, and 82; (ii) the VH CDR2 comprises the amino acid sequence of any one of SEQ ID NO: 68, 71, 74, 77, 80, and 83; (iii) the VH CDR3 comprises the amino acid sequence of any one of SEQ ID NO: 69, 72, 75, 78, 81, and 84; (iv) the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68, 71, 74, 77, 80, and 83. (v) The VL CDR2 comprises the amino acid sequence of any one of SEQ ID NO: 50, 53, 56, 59, 62, and 65; and (vi) The VL CDR3 comprises the amino acid sequence of any one of SEQ ID NO: 51, 54, 57, 60, 63, and 66; (b) a transmembrane domain; and (c) an intracellular domain comprising a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 99 or 101. In some embodiments, this disclosure provides a nucleic acid encoding an anti-CD70 CAR. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2, and VL CDR3 comprise the following amino acid sequences: (a) SEQ ID NO: 73, 74, 75, 55, 56, and 57, respectively; (b) SEQ ID NO: 67, 68, 69, 49, 50, and 51, respectively; (c) SEQ ID NO: 70, 71, 72, 52, 53, and 54, respectively; (d) SEQ ID NO: 76, 77, 78, 58, 59, and 60, respectively; (e) SEQ ID NO: 79, 80, 81, 61, 62, and 63, respectively; or (f) SEQ ID NO: 82, 83, 84, 64, 65, and 66, respectively. In some embodiments, VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2 and VL CDR3 respectively contain the following amino acid sequences: SEQ ID NO: 73, 74, 75, 55, 56 and 57.In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the following amino acid sequences: SEQ ID NO: 67, 68, 69, 49, 50, and 51. In some embodiments, VH CDR1, VHCDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the following amino acid sequences: SEQ ID NO: 70, 71, 72, 52, 53, and 54. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3 each contain the following amino acid sequences: SEQ ID NO: 76, 77, 78, 58, 59, and 60. In some embodiments, VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the following amino acid sequences: SEQ ID NO: 79, 80, 81, 61, 62, and 63. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2, and VL CDR3 each comprise the following amino acid sequences: SEQ ID NO: 82, 83, 84, 64, 65, and 66.

[0334] In some embodiments, the nucleic acid encoding the anti-CD70 CAR comprises a nucleic acid sequence of any one of SEQ ID NO: 23, 24, 26, 28, 30, 31, 33 and 35, or a nucleic acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO: 23, 24, 26, 28, 30, 31, 33 and 35.

[0335] In some embodiments, the nucleic acid encoding the anti-CD70 CAR comprises a nucleic acid sequence of any one of SEQ ID NO: 96-98 and 100, or a nucleic acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 96-98 and 100.

[0336] In some embodiments, the nucleic acid encoding the anti-CD70 CAR includes a hinge domain, wherein the hinge domain is encoded by a nucleic acid sequence of any of SEQ ID NO: 85-88 or a nucleic acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO: 85-88.

[0337] In some embodiments, the nucleic acid encoding anti-CD70 CAR comprises a nucleic acid sequence encoding a transmembrane domain, said nucleic acid sequence comprising any one of SEQ ID NO: 90-92 and 94, or a sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 90-92 and 94.

[0338] In some embodiments, the nucleic acid encoding the anti-CD70 CAR includes an activation domain, wherein the nucleic acid encoding the activation domain includes a nucleic acid sequence of SEQ ID NO: 102 or 104 or a nucleic acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 102 or 104.

[0339] In some embodiments, the nucleic acid encoding the anti-CD70 CAR comprises a nucleic acid sequence of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21, or a nucleic acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21. In some embodiments, the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, this disclosure provides mRNA encoded by the nucleic acids disclosed herein. In some embodiments, this disclosure provides expression vectors operatively linked to or comprising the nucleic acids disclosed herein.

[0340] CD70 binding protein In some implementations, the antigen-binding domain of the encoded anti-CD70 CAR includes an antibody, an antibody fragment, scFv, Fv, Fab, (Fab')2, a single-domain antibody (SDAB), a VH or VL domain, a camelid VHH domain, or a bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)).

[0341] In some cases, scFvs can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be generated by linking the VH and VL regions together using flexible peptide linkers. scFv molecules contain linkers with optimized length and / or amino acid composition (e.g., Ser-Gly linkers). Linker length can significantly influence how the variable regions of scFv fold and interact. In fact, if short peptide linkers are used (e.g., between 5-10 amino acids), intrachain folding is prevented. Interchain folding is also required to aggregate the two variable regions together to form a functional epitope binding site. Examples of connector orientation and size can be found, for example, Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, U.S. Patent Application Publications 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT Publications WO2006 / 020258 and WO2007 / 024715, which are incorporated herein by reference.

[0342] The scFv may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues between its VL and VH regions. The linker sequence may contain any naturally occurring amino acid. In some embodiments, the linker sequence contains the amino acids glycine and serine. In another embodiment, the linker sequence contains multiple sets of glycine and serine repeats, such as (Gly4Ser)n, where n is a positive integer equal to or greater than 1. In one embodiment, the linker may be (Gly4Ser)3 (SEQ ID NO: 940). Variations in linker length can preserve or enhance activity, resulting in superior efficacy in activity studies.

[0343] In some embodiments, the encoded antigen-binding domain has a binding affinity KD of 10⁻⁴ M to 10⁻⁹ M. In one embodiment, the encoded CAR molecule comprises an antigen-binding domain having a binding affinity KD of 10⁻⁴ M to 10⁻⁹ M, such as 10⁻⁵ M to 10⁻⁷ M, such as 10⁻⁶ M, or 10⁻⁷ M, such as 10⁻⁷ M to 10⁻⁸ M, such as 10⁻⁸ M to 10⁻⁹ M for the target antigen.

[0344] In one aspect, the antigen-binding domain of the anti-CD70 CAR (e.g., scFv) of this disclosure is encoded by a nucleic acid molecule whose sequence has been codon-optimized for expression in mammalian cells. In another aspect, the entire anti-CD70 CAR construct of this disclosure is encoded by a nucleic acid molecule whose entire sequence has been codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of synonymous codons (i.e., codons encoding the same amino acids) in encoding DNA varies across different species. This codon degeneracy allows the same polypeptide to be encoded by multiple nucleotide sequences.

[0345] In some embodiments, this disclosure provides an anti-CD70 chimeric antigen receptor (CAR) comprising: (a) an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises: a heavy chain variable (VH) region comprising complementarity-determining region 1 (VH CDR1), VH CDR2, and VH CDR3, and a light chain variable (VL) region comprising complementarity-determining region 1 (VL CDR1), VL CDR2, and VL CDR3, and wherein: (i) the VH CDR1 comprises the amino acid sequence of any one of SEQ ID NO: 67, 70, 73, 76, 79, and 82; (ii) the VH CDR2 comprises the amino acid sequence of any one of SEQ ID NO: 68, 71, 74, 77, 80, and 83; (iii) the VH CDR3 comprises the amino acid sequence of any one of SEQ ID NO: 69, 72, 75, 78, 81, and 84; (iv) the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68, 71, 74, 77, 80, and 83. (v) The VL CDR2 comprises the amino acid sequence of any one of SEQ ID NO: 50, 53, 56, 59, 62, and 65; and (vi) The VL CDR3 comprises the amino acid sequence of any one of SEQ ID NO: 51, 54, 57, 60, 63, and 66; (b) a transmembrane domain; and (c) an intracellular domain comprising a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 99 or 101.

[0346] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the following amino acid sequences: (a) SEQ ID NO: 73, 74, 75, 55, 56, and 57, respectively; (b) SEQ ID NO: 67, 68, 69, 49, 50, and 51, respectively; (c) SEQ ID NO: 70, 71, 72, 52, 53, and 54, respectively; (d) SEQ ID NO: 76, 77, 78, 58, 59, and 60, respectively; (e) SEQ ID NO: 79, 80, 81, 61, 62, and 63, respectively; or (f) SEQ ID NO: 82, 83, 84, 64, 65, and 66, respectively.

[0347] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the following amino acid sequences: SEQ ID NO: 73, 74, 75, 55, 56, and 57. In some embodiments, VH CDR1, VHCDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the following amino acid sequences: SEQ ID NO: 67, 68, 69, 49, 50, and 51. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3 each contain the following amino acid sequences: SEQ ID NO: 70, 71, 72, 52, 53, and 54. In some embodiments, VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the following amino acid sequences: SEQ ID NO: 76, 77, 78, 58, 59, and 60. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2, and VL CDR3 each comprise the following amino acid sequences: SEQ ID NO: 79, 80, 81, 61, 62, and 63. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the following amino acid sequences: SEQ ID NO: 82, 83, 84, 64, 65, and 66.

[0348] In some embodiments, the VH region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO: 43-48. In some embodiments, the VH region comprises an amino acid sequence of any of SEQ ID NO: 43-48, independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in each of VH CDR1, VH CDR2, and VH CDR3. In some embodiments, the VL region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO: 37-42. In some embodiments, the VL region contains the amino acid sequence of any of SEQ ID NO: 37-42, and each of VL CDR1, VL CDR2 and VL CDR3 independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.

[0349] In some embodiments, (a) the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39; (b) the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 43, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 37; (c) the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44. 38 has an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 46; (d) the VH region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 40, and the VL region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 40; (e) the VH region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 47, and the VL region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 41; or (f) the VH region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 41. 48 has an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence in SEQ ID NO: 42, and the VL region contains an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence in SEQ ID NO: 42.

[0350] In some embodiments, the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39. In some embodiments, the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 43, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 37. In some embodiments, the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 38. In some embodiments, the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 46, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 40. In some embodiments, the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 47, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 41. In some embodiments, the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 48, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 42.

[0351] In some embodiments, (a) the VH region comprises the amino acid sequence of SEQ ID NO: 45, independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in each of VH CDR1, VH CDR2, and VH CDR3, and the VL region comprises the amino acid sequence of SEQ ID NO: 39, independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in each of VL CDR1, VL CDR2, and VL CDR3; (b) the VH region comprises the amino acid sequence of SEQ ID NO: 43, independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in each of VH CDR1, VH CDR2, and VL CDR3, and the VL region comprises the amino acid sequence of SEQ ID NO: 37 ... (c) The VH region contains the amino acid sequence of SEQ ID NO: 44, each of VHCDR1, VH CDR2, and VH CDR3 independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region contains the amino acid sequence of SEQ ID NO: 38, each of VL CDR1, VL CDR2, and VL CDR3 independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions; (d) The VH region contains the amino acid sequence of SEQ ID NO: 46, each of VH CDR1, VH CDR2, and VH CDR3 independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region contains the amino acid sequence of SEQ ID NO: 48. (e) The amino acid sequence of SEQ ID NO: 47, having independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions in each of VL CDR1, VL CDR2 and VL CDR3; and the VL region contains the amino acid sequence of SEQ ID NO: 41, having independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions in each of VL CDR1, VL CDR2 and VL CDR3.Or (f) the VH region contains the amino acid sequence of SEQ ID NO: 48, independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in each of VH CDR1, VH CDR2, and VH CDR3, and the VL region contains the amino acid sequence of SEQ ID NO: 42, independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in each of VL CDR1, VL CDR2, and VL CDR3.

[0352] In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 45, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region comprises the amino acid sequence of SEQ ID NO: 39, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 43, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region comprises the amino acid sequence of SEQ ID NO: 37, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO:44, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region comprises the amino acid sequence of SEQ ID NO:38, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 46, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region comprises the amino acid sequence of SEQ ID NO: 40, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 47, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region comprises the amino acid sequence of SEQ ID NO: 41, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions.In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 48, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region comprises the amino acid sequence of SEQ ID NO: 42, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions.

[0353] In some embodiments, (a) the VH region contains the amino acid sequence of SEQ ID NO: 45 and the VL region contains the amino acid sequence of SEQ ID NO: 39; (b) the VH region contains the amino acid sequence of SEQ ID NO: 43 and the VL region contains the amino acid sequence of SEQ ID NO: 37; (c) the VH region contains the amino acid sequence of SEQ ID NO: 44 and the VL region contains the amino acid sequence of SEQ ID NO: 38; (d) the VH region contains the amino acid sequence of SEQ ID NO: 46 and the VL region contains the amino acid sequence of SEQ ID NO: 40; (e) the VH region contains the amino acid sequence of SEQ ID NO: 47 and the VL region contains the amino acid sequence of SEQ ID NO: 41; or (f) the VH region contains the amino acid sequence of SEQ ID NO: 48 and the VL region contains the amino acid sequence of SEQ ID NO: 42.

[0354] In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 45, and the VL region contains the amino acid sequence of SEQ ID NO: 39. In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 43, and the VL region contains the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 44, and the VL region contains the amino acid sequence of SEQ ID NO: 38. In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 46, and the VL region contains the amino acid sequence of SEQ ID NO: 40. In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 47, and the VL region contains the amino acid sequence of SEQ ID NO: 41. In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 48, and the VL region contains the amino acid sequence of SEQ ID NO: 42.

[0355] In some embodiments, the antigen-binding protein comprises the amino acid sequence of any one of SEQ ID NO: 25, 27, 29, 32, 34, and 36, or a sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 25, 27, 29, 32, 34, and 36. In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 27. In some embodiments, the antigen-binding protein comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 25 or SEQ ID NO: 27. In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antigen-binding protein comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 25. In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the antigen-binding protein comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 27.

[0356] In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 27, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 27, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 99.

[0357] In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 27, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 101. In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 101. In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 27, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 101.

[0358] In some embodiments, the transmembrane domain comprises a CD8a or CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain containing the amino acid sequence of SEQ ID NO: 95. In some embodiments, the transmembrane domain comprises a CD8a transmembrane region containing an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 95. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain containing the amino acid sequence of SEQ ID NO: 93. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 93.

[0359] In some embodiments, the anti-CD70 CAR further comprises a hinge domain between the antigen-binding protein and the transmembrane domain. In some embodiments, the anti-CD70 CAR comprises a linker between the anti-CD70 binding protein (e.g., scFv) and the hinge domain. In some embodiments, the linker sequence is GlySer. In some embodiments, the hinge domain is a CD8a hinge domain. In some embodiments, the hinge domain is a CD8a hinge domain or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, the hinge domain is a CD8a hinge domain or a fragment thereof comprising an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 89.

[0360] In some embodiments, the intracellular domain further comprises an activation domain. In some embodiments, the activation domain is a CD3z activation domain comprising the amino acid sequence of SEQ ID NO: 103. In some embodiments, the hinge domain is a CD3z hinge domain comprising an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 103.

[0361] In some embodiments, the intracellular domain comprises a CD28 co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 99 and a CD3z activation domain comprising the amino acid sequence of SEQ ID NO: 103. In some embodiments, the intracellular domain comprises a 41BB co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 101 and a CD3z activation domain comprising the amino acid sequence of SEQ ID NO: 103.

[0362] In some embodiments, the antigen-binding protein comprises the sequence of SEQ ID NO: 32 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; the antigen-binding protein comprises the sequence of SEQ ID NO: 32 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; the antigen-binding protein comprises the sequence of SEQ ID NO: 25 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; the antigen-binding protein comprises the sequence of SEQ ID NO: 25 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; the antigen-binding protein comprises the sequence of SEQ ID NO: 27 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; the antigen-binding protein comprises the sequence of SEQ ID NO: 27 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; the antigen-binding protein comprises the sequence of SEQ ID NO: 36 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; or the antigen-binding protein comprises the sequence of SEQ ID NO: 36 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99.

[0363] In some embodiments, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 14, 16, 20, and 22. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 2. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 4. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 16. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 20. In another embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 22.

[0364] In some implementations, the antigen-binding protein is scFv.

[0365] Preferably, the disclosed anti-CD70 CAR comprises an scFv containing CDR sequences corresponding to the light and heavy chain variable regions of the anti-CD70 antibody. Single-chain antibodies can be formed by linking heavy and light chain variable domain (Fv region) fragments via amino acid bridges (short peptide linkers) to produce a single polypeptide chain. Such single-chain Fvs (scFvs) have been prepared by fusing DNA encoding a peptide linker between DNA encoding two variable domain polypeptides (VL and VH). The resulting polypeptides can fold back to form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers) depending on the length of the flexible linker between the two variable domains (Kortt et al., 1997). Prot. Eng 10:423; Kortt et al., 2001, Biomol. Eng . 18:95-108). By combining different peptides containing VL and VH, polysaturated scFvs that bind to different epitopes can be formed (Kriangkum et al., 2001, ). Biomol. Eng . 18:31-40). Techniques developed for producing single-chain antibodies include those described in the following documents: US Patent 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544; and de Graaf et al., 2002, Methods Mol. Biol 178:379-87.

[0366] Preferably, the disclosed anti-CD70 CAR comprises an antigen-binding protein having an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any of SEQ ID NO: 25, 27, 29, 32, 34, and 36. In some embodiments, the antigen-binding protein comprises an amino acid sequence selected from any of SEQ ID NO: 25, 27, 29, 32, 34, and 36.

[0367] Hinge domain The anti-CD70 CAR described herein also includes a hinge domain. The hinge domain is located between the antigen-binding region and the transmembrane domain. A hinge domain is an amino acid segment typically found between two domains in proteins (e.g., human proteins) and, in the case of the anti-CD70 CAR, allows one or both of the antigen-binding region and the transmembrane domain to move relative to each other. Preferably, the hinge domain comprises about 10 to about 100 amino acids, for example, about 15 to about 75 amino acids, about 20 to about 50 amino acids, or about 30 to about 60 amino acids. In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein.

[0368] Preferably, the hinge domain used in the CAR is derived from CD8a. Preferably, the hinge domain of the anti-CD70 CAR comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 89. In some embodiments, the hinge domain comprises the amino acid sequence of SEQ ID NO: 89.

[0369] Preferably, the hinge domain is located between the C-terminus of the scFv and the N-terminus of the transmembrane domain of the anti-CD70 CAR.

[0370] Transmembrane domain Regarding the transmembrane domain, in various embodiments, the CAR can be designed to include a transmembrane domain attached to an extracellular domain of the CAR. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane domain, for example, one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to up to 15 amino acids from the extracellular region of the protein from which the transmembrane domain originates) and / or one or more additional amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to up to 15 amino acids from the intracellular region of the protein from which the transmembrane domain originates) are associated. In one aspect, the transmembrane domain is a transmembrane domain associated with one of the other domains of the CAR; for example, in one embodiment, the transmembrane domain may originate from the same protein as the intracellular domain, co-stimulatory domain, or hinge domain. In another aspect, the transmembrane domain does not originate from the same protein as any other domain of the CAR. In some cases, the transmembrane domain can be selectively modified, either by means of amino acid substitution, to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, for example, to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain can homodimerize with another CAR on the cell surface of a CAR-expressing cell. In other aspects, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interactions with binding domains of native binding complexes present in the same CAR-expressing cell.

[0371] The transmembrane domain can be derived from natural or recombinant sources. In the case of a natural source, the domain can originate from any membrane-binding or transmembrane protein. In one respect, whenever a CAR has bound to a target, the transmembrane domain can signal to one or more intracellular domains.

[0372] In some implementations, the transmembrane domain can be recombinant, in which case the transmembrane domain will primarily contain hydrophobic residues such as leucine and valine. In one aspect, a triplet of phenylalanine, tryptophan, and valine can be found at each end of the recombinant transmembrane domain. Optionally, short oligopeptides or polypeptide linkers with a length between 2 and 10 amino acids can form a linker between the transmembrane domain of the CAR and the cytoplasmic region. Glycine-serine duplexes provide particularly suitable linkers.

[0373] Preferably, the transmembrane domain used in the CAR is derived from membrane proteins selected from CD8a and CD28. Preferably, the transmembrane domain of the anti-CD70 CAR comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences selected from SEQ ID NO: 93 and 95. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 93 or 95.

[0374] In some embodiments, the transmembrane domain is a CD8a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the transmembrane domain is a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 93.

[0375] Intracellular domains In some embodiments of this disclosure having an intracellular domain, such a domain may contain one or more of, for example, an activation domain and / or a co-stimulatory domain. In some embodiments, the intracellular domain contains a sequence encoding an activation domain. In some embodiments, the intracellular domain contains a co-stimulatory domain. In some embodiments, the intracellular domain contains both an activation domain and a co-stimulatory domain.

[0376] The intracellular domain sequences within the cytoplasmic portion of the CAR disclosed herein can be linked together in a random or specified order. Optionally, short oligopeptides or polypeptide linkers of length, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), can form linkages between intracellular signaling sequences. In one embodiment, a glycine-serine dinucleotide can be used as a suitable linker. In one embodiment, a single amino acid (e.g., alanine, glycine) can be used as a suitable linker.

[0377] In one aspect, the intracellular domain is designed to contain two or more (e.g., 2, 3, 4, 5 or more) co-stimulatory domains. In one embodiment, the two or more (e.g., 2, 3, 4, 5 or more) co-stimulatory domains are separated by a linker molecule (e.g., the linker molecule described herein). In one embodiment, the intracellular domain contains two co-stimulatory domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0378] 1. Activate the structural domain In some embodiments, the disclosed anti-CD70 CAR includes an intracellular activation domain. Activation domains are generally responsible for the activation of at least one normal effector function of the cell. The term "effector function" describes a specific function of the cell. For example, the effector functions of T cells or NK cells include cytolytic activity or helper activity. An "activation domain" describes a protein portion that transduces effector function signals and directs the cell to perform its specific function. While the entire activation domain can be used, in many cases it is not necessary to use the entire chain or domain. Regarding the use of a truncated portion of the activation domain, such a truncated portion can be used in place of the complete domain, provided it transduces effector function signals.

[0379] Activation domains promote the activation of the TCR complex. These activation domains may contain signal transduction motifs, known as immune receptor tyrosine-based activation motifs (ITAMs). ITAM-containing activation domains for anti-CD70 CARs include intracellular domains of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Preferably, the activation domain is CD3ζ or CD28.

[0380] Preferably, the activation domain used in the CAR is derived from a membrane protein selected from CD3z. Preferably, the activation domain of the anti-CD70 CAR comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 103. In some embodiments, the activation domain comprises the amino acid sequence of SEQ ID NO: 103.

[0381] In some embodiments, the intracellular domain includes an activation domain or a functional fragment thereof, said activation domain being a CD3z activation domain comprising the amino acid sequence of SEQ ID NO: 103.

[0382] 2. Co-stimulatory domain In some embodiments, the disclosed anti-CD70 CAR includes a co-stimulatory domain. Examples of co-stimulatory domains for chimeric receptors are cytoplasmic signaling domains of co-stimulatory proteins selected from the group consisting of: members of the B7 / CD28 family (B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); and members of the TNF superfamily (4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFFR / T). NFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, Lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α and TNF RII / TNFRSF1B); members of the interleukin-1 receptor / Toll-like receptor (TLR) superfamily (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10); members of the SLAM family (2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6 and SLAM / CD150);CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLA-DR, ikaros, integrin α4 / CD49d, integrin α4 β1, integrin α4 β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP10, DAP12, MYD88, TRIF, TIRAP, TRAF, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C. Preferably, the co-stimulatory domain comprises an intracellular domain of an activating receptor protein selected from the group consisting of: α4β1 integrin, β2 integrin (CD11a-CD18, CD11b-CD18, CD11b-CD18), CD226, CRTAM, CD27, NKp46, CD16, NKp30, NKp44, NKp80, NKG2D, KIR-S, CD100, CD94 / NKG2C, CD94 / NKG2E, NKG2D, PEN5, CEACAM1, BY55, CRACC, Ly9, CD84, NTBA, 2B4, SAP, DAP10, DAP12, EAT2, FcRγ, CD3ζ, and ERT. Preferably, the co-stimulatory domain comprises an intracellular domain of an inhibitory receptor protein selected from the group consisting of: KIR-L, LILRB1, CD94 / NKG2A, KLRG-1, NKR-P1A, TIGIT, CEACAM, SIGLEC 3, SIGLEC 7, SIGLEC9, and LAIR-1. Preferably, the co-stimulatory domain comprises an intracellular domain of a protein selected from the group consisting of: CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

[0383] Preferably, the co-stimulatory domain of the anti-CD70 CAR disclosed herein is derived from a membrane protein selected from 4-1BB and CD28. Preferably, the co-stimulatory domain of the anti-CD70 CAR comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 99 or 101. In some embodiments, the co-stimulatory domain of the CAR disclosed herein comprises a 4-1BB co-stimulatory domain. In some embodiments, the co-stimulatory domain of the CAR disclosed herein comprises a CD28 co-stimulatory domain.

[0384] In some embodiments, the intracellular domain includes a co-stimulatory domain or a functional fragment thereof, said co-stimulatory domain being a 4-1BB or CD28 co-stimulatory domain. In some embodiments, the co-stimulatory domain is a 4-1BB co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 101. In some embodiments, the co-stimulatory domain is a CD28 co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 99.

[0385] Vectors containing anti-CD70 CAR In another aspect, this disclosure relates to a vector comprising a nucleic acid sequence encoding the anti-CD70CAR described herein. In one embodiment, the vector is selected from DNA vectors, RNA vectors, plastids, lentiviral vectors, adenoviral vectors, and retroviral vectors. In one embodiment, the vector is a lentiviral vector. These vectors, or portions thereof, can be used, in particular, to generate template nucleic acids as described herein for use with a CRISPR system as described herein. Alternatively, the vector can be used to deliver nucleic acids directly to cells, such as immune effector cells, such as T cells, such as allogeneic T cells, independent of the CRISPR system.

[0386] This disclosure also provides vectors in which the DNA of this disclosure is inserted. Vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow for the long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from oncogenic retroviruses (such as murine leukemia virus) because they can transduce non-proliferating cells, such as hepatocytes. Lentiviral vectors also have the added advantage of low immunogenicity. Retroviral vectors can also be, for example, gamma retroviral vectors. Gamma retroviral vectors may include, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and the transgene of interest, such as a gene encoding a CAR. Gamma retroviral vectors may lack viral structural genes, such as gag, pol, and env. Exemplary gamma retroviral vectors include murine leukemia virus (MLV), spleen focal formation virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived from said viruses. Other gamma retroviral vectors are described, for example, in Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application" Viruses. June 2011; 3(6): 677-713.

[0387] In another embodiment, the vector containing the nucleic acid encoding the desired CAR disclosed herein is an adenoviral vector (A5 / 35). In yet another embodiment, expression of the nucleic acid encoding the CAR can be achieved using transposons such as Sleeping Beauty, Crisper, CAS9, and zinc finger nucleases. See below June et al. 2009 Nature Reviews Immunology 9.10: 704-716, incorporated herein by reference.

[0388] Nucleic acids can be cloned into various types of vectors. For example, nucleic acids can be cloned into vectors, including but not limited to plasmids, phage particles, phage derivatives, animal viruses, and granules. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0389] This document discloses a method for generating in vitro transcribed RNA encoding anti-CD70 CAR. This disclosure also includes RNA constructs encoding CAR that can be directly transfected into cells. The method for generating mRNA for transfection may involve in vitro transcription (IVT) of a template using specially designed primers, followed by the addition of a poly-A tail to generate a construct containing 3' and 5' untranslated sequences (“UTR”), a 5' cap and / or an internal ribosome entry site (IRES), the nucleic acid to be expressed, and a poly-A tail typically 50–2000 bases in length. The RNA thus generated can be efficiently transfected into various cell types. In one aspect, the template includes an anti-CD70 CAR sequence.

[0390] III. Genetically modified cells containing anti-CD70 chimeric antigen receptor (CAR) and with reduced or eliminated surface expression of one or more of HLA-A, HLA-B, TRAC, MHC class II, TGFBR2, and CD70. Engineered cell compositions In another aspect, this disclosure provides engineered cells or cell populations comprising a CAR, such as the CAR described in Section II. In some embodiments, the cells are engineered to express an anti-CD70 CAR, for example, as described herein. In some embodiments, the CAR-engineered cells are allogeneic. In embodiments, the CAR-engineered cells are autologous.

[0391] In some embodiments, this disclosure provides an anti-CD70 chimeric antigen receptor (CAR) comprising: (a) an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises: a heavy chain variable (VH) region comprising complementarity-determining region 1 (VH CDR1), VH CDR2, and VH CDR3, and a light chain variable (VL) region comprising complementarity-determining region 1 (VL CDR1), VL CDR2, and VL CDR3, and wherein: (i) the VH CDR1 comprises the amino acid sequence of any one of SEQ ID NO: 67, 70, 73, 76, 79, and 82; (ii) the VH CDR2 comprises the amino acid sequence of any one of SEQ ID NO: 68, 71, 74, 77, 80, and 83; (iii) the VH CDR3 comprises the amino acid sequence of any one of SEQ ID NO: 69, 72, 75, 78, 81, and 84; (iv) the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68, 71, 74, 77, 80, and 83. (v) The VL CDR2 comprises the amino acid sequence of any one of SEQ ID NO: 50, 53, 56, 59, 62, and 65; and (vi) The VL CDR3 comprises the amino acid sequence of any one of SEQ ID NO: 51, 54, 57, 60, 63, and 66; (b) a transmembrane domain; and (c) an intracellular domain comprising a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 99 or 101.

[0392] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the following amino acid sequences: (a) SEQ ID NO: 73, 74, 75, 55, 56, and 57, respectively; (b) SEQ ID NO: 67, 68, 69, 49, 50, and 51, respectively; (c) SEQ ID NO: 70, 71, 72, 52, 53, and 54, respectively; (d) SEQ ID NO: 76, 77, 78, 58, 59, and 60, respectively; (e) SEQ ID NO: 79, 80, 81, 61, 62, and 63, respectively; or (f) SEQ ID NO: 82, 83, 84, 64, 65, and 66, respectively.

[0393] In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the following amino acid sequences: SEQ ID NO: 73, 74, 75, 55, 56, and 57. In some embodiments, VH CDR1, VHCDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the following amino acid sequences: SEQ ID NO: 67, 68, 69, 49, 50, and 51. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3 each contain the following amino acid sequences: SEQ ID NO: 70, 71, 72, 52, 53, and 54. In some embodiments, VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the following amino acid sequences: SEQ ID NO: 76, 77, 78, 58, 59, and 60. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2, and VL CDR3 each comprise the following amino acid sequences: SEQ ID NO: 79, 80, 81, 61, 62, and 63. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the following amino acid sequences: SEQ ID NO: 82, 83, 84, 64, 65, and 66.

[0394] In some embodiments, the VH region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO: 43-48. In some embodiments, the VH region comprises an amino acid sequence of any of SEQ ID NO: 43-48, independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in each of VH CDR1, VH CDR2, and VH CDR3. In some embodiments, the VL region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO: 37-42. In some embodiments, the VL region contains the amino acid sequence of any of SEQ ID NO: 37-42, and each of VL CDR1, VL CDR2 and VL CDR3 independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.

[0395] In some embodiments, (a) the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39; (b) the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 43, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 37; (c) the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44. 38 has an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 46; (d) the VH region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 40, and the VL region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 40; (e) the VH region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 47, and the VL region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 41; or (f) the VH region contains an amino acid sequence with at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 41. 48 has an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence in SEQ ID NO: 42, and the VL region contains an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence in SEQ ID NO: 42.

[0396] In some embodiments, the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39. In some embodiments, the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 43, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 37. In some embodiments, the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 38. In some embodiments, the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 46, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 40. In some embodiments, the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 47, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 41. In some embodiments, the VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 48, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 42.

[0397] In some embodiments, (a) the VH region comprises the amino acid sequence of SEQ ID NO: 45, independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in each of VH CDR1, VH CDR2, and VH CDR3, and the VL region comprises the amino acid sequence of SEQ ID NO: 39, independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in each of VL CDR1, VL CDR2, and VL CDR3; (b) the VH region comprises the amino acid sequence of SEQ ID NO: 43, independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in each of VH CDR1, VH CDR2, and VL CDR3, and the VL region comprises the amino acid sequence of SEQ ID NO: 37 ... (c) The VH region contains the amino acid sequence of SEQ ID NO: 44, each of VHCDR1, VH CDR2, and VH CDR3 independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region contains the amino acid sequence of SEQ ID NO: 38, each of VL CDR1, VL CDR2, and VL CDR3 independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions; (d) The VH region contains the amino acid sequence of SEQ ID NO: 46, each of VH CDR1, VH CDR2, and VH CDR3 independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region contains the amino acid sequence of SEQ ID NO: 48. (e) The amino acid sequence of SEQ ID NO: 47, having independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions in each of VL CDR1, VL CDR2 and VL CDR3; and the VL region contains the amino acid sequence of SEQ ID NO: 41, having independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions in each of VL CDR1, VL CDR2 and VL CDR3.Or (f) the VH region contains the amino acid sequence of SEQ ID NO: 48, independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in each of VH CDR1, VH CDR2, and VH CDR3, and the VL region contains the amino acid sequence of SEQ ID NO: 42, independently having 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in each of VL CDR1, VL CDR2, and VL CDR3.

[0398] In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 45, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region comprises the amino acid sequence of SEQ ID NO: 39, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 43, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region comprises the amino acid sequence of SEQ ID NO: 37, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO:44, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region comprises the amino acid sequence of SEQ ID NO:38, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 46, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region comprises the amino acid sequence of SEQ ID NO: 40, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 47, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region comprises the amino acid sequence of SEQ ID NO: 41, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions.In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 48, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, and the VL region comprises the amino acid sequence of SEQ ID NO: 42, each of which independently has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions.

[0399] In some embodiments, (a) the VH region contains the amino acid sequence of SEQ ID NO: 45 and the VL region contains the amino acid sequence of SEQ ID NO: 39; (b) the VH region contains the amino acid sequence of SEQ ID NO: 43 and the VL region contains the amino acid sequence of SEQ ID NO: 37; (c) the VH region contains the amino acid sequence of SEQ ID NO: 44 and the VL region contains the amino acid sequence of SEQ ID NO: 38; (d) the VH region contains the amino acid sequence of SEQ ID NO: 46 and the VL region contains the amino acid sequence of SEQ ID NO: 40; (e) the VH region contains the amino acid sequence of SEQ ID NO: 47 and the VL region contains the amino acid sequence of SEQ ID NO: 41; or (f) the VH region contains the amino acid sequence of SEQ ID NO: 48 and the VL region contains the amino acid sequence of SEQ ID NO: 42.

[0400] In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 45, and the VL region contains the amino acid sequence of SEQ ID NO: 39. In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 43, and the VL region contains the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 44, and the VL region contains the amino acid sequence of SEQ ID NO: 38. In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 46, and the VL region contains the amino acid sequence of SEQ ID NO: 40. In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 47, and the VL region contains the amino acid sequence of SEQ ID NO: 41. In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 48, and the VL region contains the amino acid sequence of SEQ ID NO: 42.

[0401] In some embodiments, the antigen-binding protein comprises the amino acid sequence of any one of SEQ ID NO: 25, 27, 29, 32, 34, and 36, or a sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 25, 27, 29, 32, 34, and 36. In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 27.

[0402] In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 27, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 27, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 99.

[0403] In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 27, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 101. In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 101. In some embodiments, the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 27, and the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 101.

[0404] In some embodiments, the transmembrane domain comprises a CD8a or CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain containing the amino acid sequence of SEQ ID NO: 95. In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 95. In some embodiments, the transmembrane domain comprises a CD28 transmembrane region containing the amino acid sequence of SEQ ID NO: 93. In some embodiments, the transmembrane domain comprises a CD28 domain having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 93.

[0405] In some embodiments, the anti-CD70 CAR further comprises a hinge domain between the antigen-binding protein and the transmembrane domain. In some embodiments, the hinge domain is a CD8a hinge domain. In some embodiments, the hinge domain is a CD8a hinge domain comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, the hinge domain is a CD8a hinge domain comprising an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 89.

[0406] In some embodiments, the intracellular domain further comprises an activation domain. In some embodiments, the activation domain is a CD3z activation domain comprising the amino acid sequence of SEQ ID NO: 103. In some embodiments, the hinge domain is a CD3z hinge domain comprising an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 103.

[0407] In some embodiments, the intracellular domain comprises a CD28 co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 99 and a CD3z activation domain comprising the amino acid sequence of SEQ ID NO: 103. In some embodiments, the intracellular domain comprises a 41BB co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 101 and a CD3z activation domain comprising the amino acid sequence of SEQ ID NO: 103.

[0408] In some embodiments, the antigen-binding protein comprises the sequence of SEQ ID NO: 32 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; the antigen-binding protein comprises the sequence of SEQ ID NO: 32 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; the antigen-binding protein comprises the sequence of SEQ ID NO: 25 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; the antigen-binding protein comprises the sequence of SEQ ID NO: 25 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; the antigen-binding protein comprises the sequence of SEQ ID NO: 27 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; the antigen-binding protein comprises the sequence of SEQ ID NO: 27 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; the antigen-binding protein comprises the sequence of SEQ ID NO: 36 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; or the antigen-binding protein comprises the sequence of SEQ ID NO: 36 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99.

[0409] In some embodiments, the anti-CD70 CAR comprises an amino acid sequence having at least 90% identity with the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 14, 16, 20, and 22. In some embodiments, the anti-CD70 CAR comprises an amino acid sequence having at least 91% identity with the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 14, 16, 20, and 22. In some embodiments, the anti-CD70 CAR comprises an amino acid sequence having at least 92% identity with the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 14, 16, 20, and 22. In some embodiments, the anti-CD70 CAR comprises an amino acid sequence having at least 93% identity with the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 14, 16, 20, and 22. In some embodiments, the anti-CD70 CAR comprises an amino acid sequence having at least 94% identity with the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 14, 16, 20, and 22. In some embodiments, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 14, 16, 20, and 22. In some embodiments, the anti-CD70 CAR comprises an amino acid sequence having at least 96% identity with the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 14, 16, 20, and 22. In some embodiments, the anti-CD70 CAR comprises an amino acid sequence having at least 97% identity with the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 14, 16, 20, and 22. In some embodiments, the anti-CD70 CAR comprises an amino acid sequence having at least 98% identity with the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 14, 16, 20, and 22. In some embodiments, the anti-CD70 CAR comprises an amino acid sequence having at least 99% identity with the amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 14, 16, 20, and 22. In some embodiments, the anti-CD70 CAR comprises an amino acid sequence of any one of SEQ ID NO: 2, 4, 6, 8, 14, 16, 20, and 22.

[0410] In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 91% identity with the amino acid sequence of SEQ ID NO: 2. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 2. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 93% identity with the amino acid sequence of SEQ ID NO: 2. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 2. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 2. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 2. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 2. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 2. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 2. In one embodiment, the anti-CD70 CAR comprises the amino acid sequence of SEQ ID NO: 2.

[0411] In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 4. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 91% identity with the amino acid sequence of SEQ ID NO: 4. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 4. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 93% identity with the amino acid sequence of SEQ ID NO: 4. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 4. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 4. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 4. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 4. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 4. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 4. In one embodiment, the anti-CD70 CAR comprises the amino acid sequence of SEQ ID NO: 4.

[0412] In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 91% identity with the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 93% identity with the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-CD70 CAR comprises the amino acid sequence of SEQ ID NO: 6.

[0413] In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 91% identity with the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 93% identity with the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-CD70 CAR comprises the amino acid sequence of SEQ ID NO: 8.

[0414] In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 91% identity with the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 93% identity with the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD70 CAR comprises the amino acid sequence of SEQ ID NO: 14.

[0415] In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 16. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 91% identity with the amino acid sequence of SEQ ID NO: 16. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 16. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 93% identity with the amino acid sequence of SEQ ID NO: 16. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 16. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 16. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 16. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 16. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 16. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 16. In one embodiment, the anti-CD70 CAR comprises the amino acid sequence of SEQ ID NO: 16.

[0416] In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 20. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 91% identity with the amino acid sequence of SEQ ID NO: 20. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 20. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 93% identity with the amino acid sequence of SEQ ID NO: 20. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 20. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 20. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 20. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 20. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 20. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 20. In one embodiment, the anti-CD70 CAR comprises the amino acid sequence of SEQ ID NO: 20.

[0417] In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 91% identity with the amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 93% identity with the amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-CD70 CAR comprises an amino acid sequence having at least 99% identity with the amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-CD70 CAR comprises the amino acid sequence of SEQ ID NO: 22.

[0418] In some implementations, the antigen-binding protein is scFv.

[0419] In some embodiments, this disclosure provides a cell population, wherein the cells are engineered cells of this disclosure.

[0420] In some aspects, the cells or cell populations disclosed herein also contain gRNA molecules as described herein, such as one or more gRNA molecules, or CRISPR systems as described herein, or gene modifications within genomic coordinates targeted by guide RNAs or CRISPR systems as described herein. In one embodiment, the cells are further altered by introducing gRNA molecules as described herein (or nucleic acids encoding said gRNA molecules) or CRISPR systems as described herein (or nucleic acids encoding one or more components of said CRISPR systems), for example, by further altering the target sequence targeted by the gRNA molecules, for example, to produce insertions / deletions, for example, by alteration using methods described herein. In one embodiment, the alteration results in reduced or absent expression of the functional (e.g., wild-type) gene product of the gene containing the target site.

[0421] In one aspect, the cell is an animal cell. In some embodiments, the cell is a mammalian, primate, or human cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune effector cell (e.g., a population of immune effector cells), such as T cells or NK cells. In embodiments, the T cell (e.g., a population of T cells) is or comprises CD4+ T cells, CD8+ T cells, or a combination thereof. In some embodiments, the cell is a human T cell, such as a human T cell or a population of human T cells. In some embodiments, the cell is a population of human T cells comprising cells expressing CD4 and / or CD8. In embodiments, the cell or cell population is autologous. In embodiments, the cell or cell population is allogeneic.

[0422] In another aspect, this disclosure further provides cells, such as those described above, including a second gRNA molecule as described herein, for example, a second gRNA molecule having a guide sequence different from that of the first gRNA molecule. In other embodiments, two or more gRNA molecules are complementary to target sites within two different genes that associate with gene products to form molecular complexes. It will be understood that in any aspect and embodiment of this disclosure targeting two or more target sites of different genes (or different molecular complexes, e.g., when targeting TCR and HLA-A), two or more gRNAs may be employed for any or all of the different gene (or molecular complex) targets, with respect to one or more of the different genes or different molecular complexes. For example, in embodiments and aspects of reduced or eliminated expression of TCR and HLA-A, reduced or eliminated expression of TCR can be achieved, for example, by one gRNA targeting TRAC or by more than one gRNA molecule targeting TRAC; simultaneously or alternatively, targeting of HLA-A can be achieved, for example, by one gRNA molecule targeting HLA-A or by two or more gRNA molecules targeting HLA-A. In other embodiments, two or more (e.g., two) gRNA molecules are complementary to target sites within different genes. Such cells may contain alterations, such as insertions / deletions, at or near each target site, resulting in reduced or eliminated expression of the functional gene product of more than one gene. As discussed above, in such embodiments, more than one gRNA molecule targeting each of the different genes may be employed.

[0423] In one embodiment, the cell contains one or more gRNA molecules, the gRNA molecules containing a guide sequence complementary to a target sequence of HLA-A, HLA-B, TRAC, CIITA, TGFBR2, or CD70.

[0424] In embodiments, this disclosure provides a cell, such as a cell containing a CAR (e.g., as described herein), said cell containing one or more modifications (e.g., nucleotide insertions or deletions) to an endogenous gene encoding HLA-A, HLA-B, TRAC, CIITA, TGFBR2, and / or CD70.

[0425] In some embodiments, the engineered cells further comprise reduced or eliminated TGFBR2 surface expression relative to unmodified cells, the engineered cells comprising a genetic modification of the TGFBR2 gene, wherein the genetic modification comprises at least one nucleotide within the following genomic coordinates: chr3:30606864-30691614. In some embodiments, the engineered cells have reduced or eliminated TGFBR2 surface expression relative to unmodified cells, the engineered cells comprising a genetic modification of the TGFBR2 gene, wherein the genetic modification is within the following genomic coordinates: 30606891-30691605. In some embodiments, the engineered cells have reduced or eliminated TGFBR2 surface expression relative to unmodified cells, the engineered cells comprising a genetic modification of the TGFBR2 gene, wherein the genetic modification is within the genomic coordinates chr3:30674205-30674229. In some embodiments, the engineered cells have reduced or eliminated TGFBR2 surface expression relative to unmodified cells, the engineered cells comprising a genetic modification of the TGFBR2 gene, wherein the genetic modification comprises at least one nucleotide within genomic coordinates targeted by a TGFBR2 guide RNA comprising the guide sequence of SEQ ID NO: SEQ ID NO: 301. In some embodiments, the engineered cells have reduced or eliminated TGFBR2 surface expression relative to unmodified cells, the engineered cells comprising a genetic modification of the TGFBR2 gene, wherein the genetic modification is within the following genomic coordinates: chr3:30606864-30691614. In some embodiments, the engineered cells have reduced or eliminated CD70 surface expression relative to unmodified cells, the engineered cells comprising a genetic modification of the CD70 gene, wherein the genetic modification is within the genomic coordinates chr19:6586002-6591015. In some embodiments, the genetic modification within the genomic coordinates comprises insertions / deletions, C-to-T substitutions, or A-to-G substitutions. In some embodiments, the gene modification includes C-to-T substitutions within the genomic coordinate system. In some embodiments, the cells are homozygous for HLA-C. In some embodiments, the cells are homozygous for both HLA-B and HLA-C.

[0426] In some embodiments, the engineered cells have reduced or eliminated CD70 surface expression relative to unmodified cells, the engineered cells comprising a genetic modification of the CD70 gene, wherein the genetic modification comprises at least one nucleotide within genomic coordinates targeted by a CD70 guide RNA comprising the guide sequence of SEQ ID NO: 310. In some embodiments, the engineered cells have reduced or eliminated CD70 surface expression relative to unmodified cells, the engineered cells comprising a genetic modification of the CD70 gene, wherein the genetic modification comprises at least one nucleotide within genomic coordinates chr19: 6586028-6591018. In some embodiments, the engineered cells have reduced or eliminated CD70 surface expression relative to unmodified cells, the engineered cells comprising a genetic modification of the CD70 gene, wherein the genetic modification comprises at least one nucleotide within genomic coordinates chr19: 6590998-6591018. In some embodiments, the engineered cells have reduced or eliminated CD70 surface expression relative to unmodified cells, said engineered cells containing a genetic modification of the CD70 gene, wherein said genetic modification comprises at least one nucleotide within a genomic coordinate system targeted by a CD70 guide RNA comprising the guide sequence of SEQ ID NO: 312. In some embodiments, the genetic modification comprises insertion / deletion, C-to-T substitution, or A-to-G substitution within the genomic coordinate system. In some embodiments, the genetic modification comprises C-to-T substitution within the genomic coordinate system. In some embodiments, the cells are homozygous for HLA-C. In some embodiments, the cells are homozygous for both HLA-B and HLA-C.

[0427] In some embodiments, the engineered cells have reduced or eliminated surface expression of TGFBR2 and CD70 compared to unmodified cells, and the engineered cells contain gene modifications in the TGFB2 and CD70 genes. In some embodiments, the cells are homozygous for HLA-C. In some embodiments, the cells are homozygous for both HLA-B and HLA-C.

[0428] In some embodiments of this disclosure, the engineered cells further comprise reduced or eliminated HLA-A surface expression relative to unmodified cells, the engineered cells comprising gene modifications in the HLA-A gene, wherein the gene modifications are located within gene coordinates chr6: 29942540-29945459. In some embodiments, the engineered cells have reduced or eliminated HLA-A surface expression relative to unmodified cells, the engineered cells containing a genetic modification in the HLA-A gene, wherein the genetic modification is located within the following genomic coordinates: chr6:29942891-29942915; chr6:29942609-29942633; chr6:29942889-29942913; chr6:29944471-29944495; chr6:29944266-29944290; and chr6:29942785-29942809. In some embodiments, the engineered cells have reduced or eliminated HLA-A surface expression relative to unmodified cells, said engineered cells containing a genetic modification in the HLA-A gene, wherein said genetic modification comprises at least one nucleotide within a genomic coordinate system targeted by an HLA-A guide RNA comprising the guide sequence of SEQ ID NO: 403. In some embodiments, the genetic modification comprises insertion / deletion, C-to-T substitution, or A-to-G substitution within the genomic coordinate system. In some embodiments, the genetic modification comprises C-to-T substitution within the genomic coordinate system. In some embodiments, the cells are homozygous for HLA-C. In some embodiments, the cells are homozygous for both HLA-B and HLA-C.

[0429] In some embodiments, the engineered cells further have reduced or eliminated HLA-B surface expression relative to unmodified cells, said engineered cells containing a genetic modification in the HLA-B gene, wherein said genetic modification contains at least one nucleotide within the genomic coordinates chr6:31354480-31357174. In some embodiments, the engineered cells have reduced or eliminated HLA-B surface expression relative to unmodified cells, said engineered cells containing a genetic modification in the HLA-B gene, wherein said genetic modification is within the following genomic coordinates: chr6:31355222-31355246, chr6:31355221-31355245, or chr6:31355205-31355229. In some embodiments, the engineered cells have reduced or eliminated HLA-B surface expression relative to unmodified cells, said engineered cells containing a genetic modification in the HLA-B gene, wherein said genetic modification comprises at least one nucleotide within a genomic coordinate system targeted by an HLA-B guide RNA comprising the guide sequence of SEQ ID NO: 405-407. In some embodiments, the genetic modification comprises insertion / deletion, C-to-T substitution, or A-to-G substitution within the genomic coordinate system. In some embodiments, the genetic modification comprises C-to-T substitution within the genomic coordinate system. In some embodiments, the cells are homozygous for HLA-C.

[0430] In some embodiments, the engineered cells further have reduced or eliminated TRAC surface expression relative to unmodified cells, the engineered cells containing a genetic modification in the TRAC gene, wherein the genetic modification contains at least one nucleotide within genomic coordinates chr14:22547462-22551621. In some embodiments, the engineered cells have reduced or eliminated TRAC surface expression relative to unmodified cells, the engineered cells containing a genetic modification in the TRAC gene, wherein the genetic modification contains at least one nucleotide within genomic coordinates chr14:22547524-22547544. In some embodiments, the engineered cells have reduced or eliminated TRAC surface expression relative to unmodified cells, the engineered cells containing a genetic modification in the TRAC gene, wherein the genetic modification contains at least one nucleotide within genomic coordinates targeted by a TRAC guide RNA containing the guide sequence of SEQ ID NO: 413. In some embodiments, the genetic modification within genomic coordinates includes insertions / deletions, C-to-T substitutions, or A-to-G substitutions. In some embodiments, the genetic modification within genomic coordinates includes C-to-T substitutions. In some implementations, the cells are homozygous for HLA-C. In some implementations, the cells are homozygous for both HLA-B and HLA-C.

[0431] In some embodiments, the engineered cells further have reduced or eliminated MHC class II surface expression relative to unmodified cells, said engineered cells containing a genetic modification in the CIITA gene, said genetic modification being located within a genomic coordinate system selected from the following: (a) chr16:10877363-10907788 and (b) chr16:10906515-10908136. In some embodiments, the engineered cells have reduced or eliminated MHC class II surface expression relative to unmodified cells, said engineered cells containing a genetic modification in the CIITA gene, said genetic modification being located within a genomic coordinate system targeted by a CIITA guide RNA containing the guide sequence of SEQ ID NO: 401. In some embodiments, the genetic modification is located within a genomic coordinate system containing insertions / deletions, C-to-T substitutions, or A-to-G substitutions. In some embodiments, the genetic modification is located within a genomic coordinate system containing C-to-T substitutions. In some embodiments, the cells are homozygous for HLA-C. In some implementations, the cells are homozygous for both HLA-B and HLA-C.

[0432] In some embodiments, the engineered cells have reduced or eliminated surface expression of HLA-A, TRAC, and MHC class II compared to unmodified cells, and the engineered cells contain gene modifications in the HLA-A, TRAC, and CIITA genes. In some embodiments, the cells are homozygous for HLA-C. In some embodiments, the cells are homozygous for both HLA-B and HLA-C.

[0433] In some embodiments, the engineered cells have reduced or eliminated surface expression of HLA-A, HLA-B, TRAC, and MHC class II compared to unmodified cells, and the engineered cells contain gene modifications in the HLA-A, HLA-B, TRAC, and CIITA genes. In some embodiments, the cells are homozygous for HLA-C.

[0434] In some embodiments, the engineered cells have reduced or eliminated surface expression of HLA-A, TRAC, MHC class II, and TGFBR2 compared to unmodified cells, and the engineered cells contain gene modifications in the HLA-A, TRAC, CIITA, and TGFBR2 genes. In some embodiments, the cells are homozygous for HLA-C. In some embodiments, the cells are homozygous for both HLA-B and HLA-C.

[0435] In some embodiments, the engineered cells have reduced or eliminated surface expression of HLA-A, HLA-B, TRAC, MHC class II, and TGFBR2 compared to unmodified cells, and the engineered cells contain gene modifications in the HLA-A, HLA-B, TRAC, CIITA, and TGFBR2 genes.

[0436] In some implementations, the cells are homozygous for HLA-C.

[0437] In some embodiments, the engineered cells have reduced or eliminated surface expression of HLA-A, TRAC, MHC class II, and CD70 compared to unmodified cells, and the engineered cells contain gene modifications in the HLA-A, TRAC, CIITA, and CD70 genes. In some embodiments, the cells are homozygous for HLA-C. In some embodiments, the cells are homozygous for both HLA-B and HLA-C.

[0438] In some embodiments, the engineered cells have reduced or eliminated surface expression of HLA-A, HLA-B, TRAC, MHC class II, and CD70 compared to unmodified cells, and the engineered cells contain gene modifications in the HLA-A, HLA-B, TRAC, CIITA, and CD70 genes. In some embodiments, the cells are homozygous for HLA-C.

[0439] In some embodiments, the engineered cells have reduced or eliminated surface expression of HLA-A, TRAC, MHC class II, TGFBR2, and CD70 compared to unmodified cells, and the engineered cells contain gene modifications in the HLA-A, TRAC, CIITA, TGFBR2, and CD70 genes. In some embodiments, the cells are homozygous for HLA-C. In some embodiments, the cells are homozygous for both HLA-B and HLA-C.

[0440] In some embodiments, the engineered cells have reduced or eliminated surface expression of HLA-A, HLA-B, TRAC, MHC class II, TGFBR2, and CD70 compared to unmodified cells, and the engineered cells contain gene modifications in the HLA-A, TRAC, CIITA, and CD70 genes. In some embodiments, the cells are homozygous for HLA-C.

[0441] In some embodiments, this disclosure provides an engineered cell comprising a modified HLA-A gene, a modified TRAC gene, a modified CIITA gene, a modified TGFBR2 gene, and / or a modified CD70 gene, wherein the engineered cell expresses an anti-CD70 chimeric antigen receptor (CAR) or contains nucleic acid encoding an anti-CD70 CAR. In some embodiments, the cell is homozygous for HLA-C. In some embodiments, the cell is homozygous for both HLA-B and HLA-C.

[0442] In some embodiments, the gene modification in TGFBR2 contains at least one nucleotide within the following genomic coordinates: chr3:30606864-30691614. In some embodiments, the gene modification in TGFBR2 contains the following genomic coordinates: 30606891-30691605. In some embodiments, the gene modification in TGFBR2 contains the following genomic coordinates: chr3:30674205-30674229. In some embodiments, the gene modification in TGFBR2 contains at least one nucleotide within the genomic coordinates targeted by the TGFBR2 guide RNA containing the guide sequence of SEQ ID NO: 301. In some embodiments, the gene modification in TGFBR2 contains the following genomic coordinates: chr3:30606864-30691614. In some embodiments, the gene modification in TGFBR2 contains at least one nucleotide within the genomic coordinates targeted by the TGFBR2 guide RNA containing the guide sequence of SEQ ID NO: 302. In some embodiments, the gene modification includes insertions / deletions, C-to-T substitutions, or A-to-G substitutions within genomic coordinates. In some embodiments, the gene modification includes C-to-T substitutions within genomic coordinates.

[0443] In some embodiments, the gene modification in CD70 is located within the genomic coordinates chr19:6586002-6591015. In some embodiments, the gene modification in CD70 contains at least one nucleotide within the genomic coordinates targeted by a CD70 guide RNA containing the guide sequence of SEQ ID NO: 310. In some embodiments, the gene modification in CD70 is located within the following genomic coordinates: chr19: 6586028-6591018. In some embodiments, the gene modification in CD70 contains at least one nucleotide within the genomic coordinates chr19:6590998-6591018. In some embodiments, the gene modification in CD70 contains at least one nucleotide within the genomic coordinates targeted by a CD70 guide RNA containing the guide sequence of SEQ ID NO: 312. In some embodiments, the gene modification contains insertions / deletions, C-to-T substitutions, or A-to-G substitutions within the genomic coordinates. In some embodiments, the gene modification contains C-to-T substitutions within the genomic coordinates.

[0444] In some embodiments, the gene modification in HLA-A is located within the genomic coordinates chr6: 29942540-29945459. In some embodiments, the gene modification in HLA-A is located within the genomic coordinates selected from the following: chr6:29942891-29942915; chr6:29942609-29942633; chr6:29942889-29942913; chr6:29944471-29944495; chr6:29944266-29944290; and chr6:29942785-29942809. In some embodiments, the gene modification in HLA-A contains at least one nucleotide within the genomic coordinates targeted by the HLA-A guide RNA containing the guide sequence of SEQ ID NO: 403. In some embodiments, the gene modification includes insertions / deletions, C-to-T substitutions, or A-to-G substitutions within genomic coordinates. In some embodiments, the gene modification includes C-to-T substitutions within genomic coordinates.

[0445] In some embodiments, the gene modification in the TRAC contains at least one nucleotide within the genomic coordinates chr14:22547462-22551621. In some embodiments, engineered cells have reduced or eliminated surface expression of TRAC relative to unmodified cells, said engineered cells containing the gene modification in the TRAC gene, wherein said gene modification is within the genomic coordinates chr14:22547524-22547544. In some embodiments, the gene modification in the TRAC contains at least one nucleotide within the genomic coordinates targeted by a TRAC guide RNA containing the guide sequence of SEQ ID NO: 413. In some embodiments, the gene modification contains insertions / deletions, C-to-T substitutions, or A-to-G substitutions within the genomic coordinates. In some embodiments, the gene modification contains C-to-T substitutions within the genomic coordinates.

[0446] In some embodiments, the genetic modification in CIITA is located within the genomic coordinates selected from the following: (a) chr16:10877363-10907788 and (b) chr16:10906515-10908136. In some embodiments, the engineered cells have reduced or eliminated surface expression of CIITA relative to unmodified cells, said engineered cells containing the genetic modification in the CIITA gene, said genetic modification being located within the genomic coordinates selected from the following: chr16:10906643-10906667 and chr16:10907504-10907528. In some embodiments, the genetic modification in CIITA contains at least one nucleotide within the genomic coordinates targeted by a CIITA guide RNA containing the guide sequence of SEQ ID NO: 402 or 401. In some embodiments, the genetic modification contains insertions / deletions, C-to-T substitutions, or A-to-G substitutions within the genomic coordinates. In some implementations, the gene modification includes C to T substitutions within the genomic coordinate system.

[0447] In some embodiments, this disclosure provides an engineered cell comprising a modified HLA-A gene, a modified HLA-B gene, a modified TRAC gene, a modified CIITA gene, a modified TGFBR2 gene, and / or a modified CD70 gene, wherein the engineered cell expresses an anti-CD70 chimeric antigen receptor (CAR) or contains nucleic acid encoding an anti-CD70 CAR. In some embodiments, the cell is homozygous for HLA-C.

[0448] In some embodiments, the gene modification in TGFBR2 contains at least one nucleotide within the following genomic coordinates: chr3:30606864-30691614. In some embodiments, the gene modification in TGFBR2 contains the following genomic coordinates: 30606891-30691605. In some embodiments, the gene modification in TGFBR2 contains the following genomic coordinates: chr3:30674205-30674229. In some embodiments, the gene modification in TGFBR2 contains at least one nucleotide within the genomic coordinates targeted by the TGFBR2 guide RNA containing the guide sequence of SEQ ID NO: 301. In some embodiments, the gene modification in TGFBR2 contains the following genomic coordinates: chr3:30606864-30691614. In some embodiments, the gene modification in TGFBR2 contains at least one nucleotide within the genomic coordinates targeted by the TGFBR2 guide RNA containing the guide sequence of SEQ ID NO: 302. In some embodiments, the gene modification includes insertions / deletions, C-to-T substitutions, or A-to-G substitutions within genomic coordinates. In some embodiments, the gene modification includes C-to-T substitutions within genomic coordinates.

[0449] In some embodiments, the gene modification in CD70 is located within the genomic coordinates chr19:6586002-6591015. In some embodiments, the gene modification in CD70 contains at least one nucleotide within the genomic coordinates targeted by a CD70 guide RNA containing the guide sequence of SEQ ID NO: 310. In some embodiments, the gene modification in CD70 is located within the following genomic coordinates: chr19:6586028-6591018. In some embodiments, the gene modification in CD70 contains at least one nucleotide within the genomic coordinates chr19:6590998-6591018. In some embodiments, the gene modification in CD70 contains at least one nucleotide within the genomic coordinates targeted by a CD70 guide RNA containing the guide sequence of SEQ ID NO: 312. In some embodiments, the gene modification contains insertions / deletions, C-to-T substitutions, or A-to-G substitutions within the genomic coordinates. In some embodiments, the gene modification contains C-to-T substitutions within the genomic coordinates.

[0450] In some embodiments, the gene modification in HLA-A is located within the genomic coordinates chr6: 29942540-29945459. In some embodiments, the gene modification in HLA-A is located within the genomic coordinates selected from the following: chr6:29942891-29942915; chr6:29942609-29942633; chr6:29942889-29942913; chr6:29944471-29944495; chr6:29944266-29944290; and chr6:29942785-29942809. In some embodiments, the gene modification in HLA-A contains at least one nucleotide within the genomic coordinates targeted by the HLA-A guide RNA containing the guide sequence of SEQ ID NO: 403. In some embodiments, the gene modification includes insertions / deletions, C-to-T substitutions, or A-to-G substitutions within genomic coordinates. In some embodiments, the gene modification includes C-to-T substitutions within genomic coordinates.

[0451] In some embodiments, the gene modification in HLA-B contains at least one nucleotide within the genomic coordinates chr6:31354480-31357174. In some embodiments, the gene modification in HLA-B contains at least one nucleotide within the following genomic coordinates: chr6:31355222-31355246, chr6:31355221-31355245, or chr6:31355205-31355229. In some embodiments, the gene modification in HLA-B contains at least one nucleotide within the genomic coordinates targeted by an HLA-B guide RNA containing the guide sequence of SEQ ID NO: 405-407. In some embodiments, the gene modification contains insertions / deletions, C-to-T substitutions, or A-to-G substitutions within the genomic coordinates. In some embodiments, the gene modification contains C-to-T substitutions within the genomic coordinates.

[0452] In some embodiments, the gene modification in the TRAC contains at least one nucleotide within the genomic coordinates chr14:22547462-22551621. In some embodiments, engineered cells have reduced or eliminated surface expression of TRAC relative to unmodified cells, said engineered cells containing the gene modification in the TRAC gene, wherein said gene modification is within the genomic coordinates chr14:22547524-22547544. In some embodiments, the gene modification in the TRAC contains at least one nucleotide within the genomic coordinates targeted by a TRAC guide RNA containing the guide sequence of SEQ ID NO: 413. In some embodiments, the gene modification contains insertions / deletions, C-to-T substitutions, or A-to-G substitutions within the genomic coordinates. In some embodiments, the gene modification contains C-to-T substitutions within the genomic coordinates.

[0453] In some embodiments, the genetic modification in CIITA is located within the genomic coordinates selected from the following: (a) chr16:10877363-10907788 and (b) chr16:10906515-10908136. In some embodiments, the engineered cells have reduced or eliminated surface expression of CIITA relative to unmodified cells, said engineered cells containing the genetic modification in the CIITA gene, said genetic modification being located within the genomic coordinates selected from the following: chr16:10906643-10906667 and chr16:10907504-10907528. In some embodiments, the genetic modification in CIITA contains at least one nucleotide within the genomic coordinates targeted by a CIITA guide RNA containing the guide sequence of SEQ ID NO: 402. In some embodiments, the genetic modification in CIITA contains at least one nucleotide within the genomic coordinates targeted by a CIITA guide RNA containing the guide sequence of SEQ ID NO: 401. In some embodiments, the gene modification includes insertions / deletions, C-to-T substitutions, or A-to-G substitutions within genomic coordinates. In some embodiments, the gene modification includes C-to-T substitutions within genomic coordinates.

[0454] In some embodiments of this disclosure, the gene modification comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive nucleotides within genomic coordinates. In some embodiments, the gene modification comprises insertion / deletion. In some embodiments, the gene modification comprises insertion of a heterologous coding sequence. In some embodiments, the gene modification comprises at least one A to G substitution within genomic coordinates. In some embodiments, the gene modification comprises at least one C to T substitution within genomic coordinates.

[0455] In some embodiments, the cells have reduced expression of the TRAC protein on the cell surface. In some embodiments, the cells have genetic modifications in the CIITA gene. In some embodiments, the cells have reduced expression of MHC class II molecules on the cell surface.

[0456] In some embodiments, this disclosure provides a cell population comprising the engineered cells of this disclosure.

[0457] In some embodiments, this disclosure provides a pharmaceutical composition comprising the engineered cells of this disclosure.

[0458] In some aspects, this disclosure provides engineered cells, cell populations, pharmaceutical compositions, or methods, wherein the engineered cells are immune cells.

[0459] In some aspects, this disclosure provides engineered cells, cell populations, pharmaceutical compositions, or methods, wherein the engineered cells are stem cells.

[0460] In some aspects, this disclosure provides engineered cells, cell populations, pharmaceutical compositions, or methods, wherein the engineered cells are primary cells.

[0461] In some aspects, this disclosure provides engineered cells, cell populations, pharmaceutical compositions, or methods, wherein the engineered cells are engineered using a genome editing system. In some embodiments, the genome editing system comprises an RNA-guided DNA binder or a nucleic acid encoding the RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder or the nucleic acid-encoded RNA-guided DNA binder is *Streptococcus pyogenes* Cas9 (SpyCas9). In some embodiments, the RNA-guided DNA binder or the nucleic acid-encoded RNA-guided DNA binder is *Neisseria meningitidis* Cas9 (NmeCas9). In some embodiments, the RNA-guided DNA binder or the nucleic acid-encoded RNA-guided DNA binder has double-stranded endonuclease activity. The RNA-guided DNA binder or the nucleic acid-encoded RNA-guided DNA binder has nicking enzyme activity. In some embodiments, the RNA-guided DNA binder or the nucleic acid-encoded RNA-guided DNA binder comprises a dCas9 DNA-binding domain. In some embodiments, the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is an A-G base editor. In some implementations, the RNA-guided DNA binder or the nucleic acid encoding the RNA-guided DNA binder is a C-to-T base editor.

[0462] In some embodiments, the guide RNA is provided to the cells in a vector. In some embodiments, an RNA-guided DNA binder is provided to the cells in a vector, optionally in the same vector as the guide RNA. In some embodiments, a foreign nucleic acid is provided to the cells in a vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector.

[0463] In some embodiments, the guide RNA is provided to the cell in a lipid nanoparticle (LNP), optionally in the same LNP that provides an RNA-guided DNA binder. In some embodiments, the exogenous nucleic acid is provided to the cell in a lipid nanoparticle (LNP). In some embodiments, the guide gRNA is a single guide RNA. In some embodiments, the guide RNA contains a 5' end modification or a 3' end modification.

[0464] In the implementation scheme, the modification reduces or eliminates the expression of the gene. In embodiments, this disclosure provides a cell, such as a cell containing a CAR (e.g., as described herein), said cell being HLA-A- (e.g., having HLA-A expression levels that are 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower than those of unmodified cells of the same type, as detected by FACS, such as FACS using an anti-HLA-A antibody), HLA-B- (e.g., having HLA-B expression levels that are 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower than those of unmodified cells of the same type, as detected by FACS, such as FACS using an anti-HLA-B antibody), TCR- (e.g., having TCR expression levels that are 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower than those of unmodified cells of the same type, as detected by FACS, such as FACS using an anti-CD3 antibody), CIITA- (e.g., having CIITA and / or MHC class II protein expression levels that are 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower than those of unmodified cells of the same type, as detected by FACS, such as FACS using an anti-CIITA antibody), TGFBR2- (e.g., having TGFBR2 expression levels that are 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower than those of unmodified cells of the same type, as detected by FACS, such as FACS using an anti-TGFBR2 antibody), and / or CD70- (e.g., having CD70 expression levels that are 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower than those of unmodified cells of the same type, as detected by FACS, such as FACS using an anti-CD70 antibody).

[0465] In some embodiments of this disclosure, the cells or cell populations of this disclosure comprise: (a) a nucleic acid sequence encoding a CAR, such as that disclosed herein; and (b) an insertion / deletion at or near a sequence of a gene encoding one or more of HLA-A, HLA-B, TRAC, CIITA, TGFBR2, and CD70, or a regulatory element thereof, such as an insertion / deletion at or near a target sequence of a gRNA containing a guide sequence (e.g., containing guide sequences listed in Tables 2A-2B) targeting one or more of HLA-A, HLA-B, TRAC, CIITA, TGFBR2, and CD70.

[0466] In any of the embodiments and aspects, the cell may also comprise one or more CRISPR systems containing one or more indicated gRNA molecules, for example, as described herein. In some embodiments, the cell comprises one or more ribonucleoprotein (RNP) complexes, each RNP complex containing a Cas9 molecule (e.g., as described herein) and a gRNA molecule containing an indicated guide sequence (e.g., as described herein). In some embodiments, including any of the methods described herein that employ gRNAs targeting more than one target sequence, the gRNA (and the CRISPR system containing the gRNA) may be introduced into the cell simultaneously. In other embodiments, including any of the methods described herein that employ gRNAs targeting more than one target sequence, the gRNA (and the CRISPR system containing the gRNA) may be introduced into the cell sequentially.

[0467] In any of the foregoing embodiments or aspects, the cell population comprises at least 20%, such as at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, cells comprising insertions / deletions at or near each of the target sequences targeted by each of the gRNA molecules. The population can be obtained, for example, by utilizing highly efficient gRNA molecules (e.g., gRNA molecules that induce insertions / deletions in cells >85% exposed to said gRNA molecules) or by enriching the population with desired cells, for example, by selecting the desired cell population, such as by affinity chromatography or cell sorting.

[0468] Exemplary engineered cell compositions In addition to providing engineered cells or cell populations as described in the preceding subsections, this disclosure also provides engineered cells, cell populations, pharmaceutical compositions, and uses thereof.

[0469] In some embodiments, this document provides an engineered human T cell comprising multiple genetic modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein (a) the engineered human T cell comprises genetic modification of the HLA-A gene and reduced or eliminated HLA-A surface expression relative to unmodified cells, genetic modification of the HLA-B gene and reduced or eliminated HLA-B surface expression relative to unmodified cells, genetic modification of the CIITA gene and reduced or eliminated MHC class II surface expression relative to unmodified cells, genetic modification of the TGFBR2 gene and reduced or eliminated TGFBR2 surface expression relative to unmodified cells, and genetic modification of the CD70 gene and reduced or eliminated CD70 surface expression relative to unmodified cells, and (b) the anti-CD70 CAR comprises an antigen-binding protein or fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region, the heavy chain variable region comprising a complementarity-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, and comprising the amino acid sequence of SEQ ID NO: 73. The anti-CD70 CAR contains a VH CDR2 containing the amino acid sequence of SEQ ID NO: 74 and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 75, and contains a light chain variable (VL) region comprising a complementarity-determining region 1 (VL CDR1) containing the amino acid sequence of SEQ ID NO: 55, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 56, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted into the TRAC locus.

[0470] In some embodiments, this document provides an engineered human T cell comprising multiple genetic modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein (a) the engineered human T cell comprises genetic modifications within genomic coordinates chr6:29942891-29942915 of the HLA-A gene, genetic modifications within genomic coordinates chr6:31355222-31355246 of the HLA-B gene, genetic modifications within genomic coordinates chr16:10906643-10906667 of the CIITA gene, genetic modifications within genomic coordinates chr3:30674205-30674229 of the TGFBR2 gene, and genetic modifications within genomic coordinates chr19:6590121-6590145 of the CD70 gene, and (b) the anti-CD70... The CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region comprising a complementation-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 75, and a light chain variable (VL) region comprising a complementation-determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 55, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted into the TRAC locus.

[0471] In some embodiments, this document provides an engineered human T cell comprising multiple genetic modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein (a) the engineered human T cell comprises genetic modifications within genomic coordinates chr6:29942891-29942915 of the HLA-A gene, genetic modifications within genomic coordinates chr6:31355222-31355246 of the HLA-B gene, genetic modifications within genomic coordinates chr16:10906643-10906667 of the CIITA gene, genetic modifications within genomic coordinates chr3:30674205-30674229 of the TGFBR2 gene, and genetic modifications within genomic coordinates chr19:6590121-6590145 of the CD70 gene, and (b) the anti-CD70... The CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region, the heavy chain variable region comprising a complementation-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 75, and a light chain variable (VL) region, the light chain variable region comprising a complementation-determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 55, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted at genomic coordinates chr14:22547524-22547544 at the TRAC locus.

[0472] In some embodiments, this document provides a pharmaceutical composition comprising a population of T cells comprising CD4+ T cells and / or CD8+ T cells, the cells comprising engineered human T cells comprising multiple genetic modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein (a) the engineered human T cells comprise genetic modifications to the HLA-A gene and reduced or eliminated HLA-A surface expression relative to unmodified cells, genetic modifications to the HLA-B gene and reduced or eliminated HLA-B surface expression relative to unmodified cells, genetic modifications to the CIITA gene and reduced or eliminated MHC class II surface expression relative to unmodified cells, genetic modifications to the TGFBR2 gene and reduced or eliminated TGFBR2 surface expression relative to unmodified cells, and genetic modifications to the CD70 gene and reduced or eliminated CD70 surface expression relative to unmodified cells, and (b) the anti-CD70 CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region, the heavy chain variable region comprising SEQ ID NO: The anti-CD70 CAR contains a complementation-determining region 1 (VH CDR1) of the amino acid sequence of SEQ ID NO: 73, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 containing the amino acid sequence of SEQ ID NO: 75, and contains a light chain variable (VL) region comprising a complementation-determining region 1 (VLCDR1) containing the amino acid sequence of SEQ ID NO: 55, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 56, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted into the TRAC locus.

[0473] In some embodiments, this document provides a T cell population comprising CD4+ T cells and / or CD8+ T cells, the cells comprising engineered human T cells containing multiple genetic modifications and anti-CD70 chimeric antigen receptor (CAR), wherein (a) the engineered human T cells contain genetic modifications within genomic coordinates chr6:29942891-29942915 of the HLA-A gene, genetic modifications within genomic coordinates chr6:31355222-31355246 of the HLA-B gene, genetic modifications within genomic coordinates chr16:10906643-10906667 of the CIITA gene, genetic modifications within genomic coordinates chr3:30674205-30674229 of the TGFBR2 gene, and genetic modifications within genomic coordinates chr19:6590121-6590145 of the CD70 gene, and (b) the anti-CD70... The CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region, the heavy chain variable region comprising a complementarity-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 75, and a light chain variable (VL) region, the light chain variable region comprising a complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence of SEQ ID NO: 55, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted into the TRAC locus.

[0474] In some embodiments, this document provides a T cell population comprising CD4+ T cells and / or CD8+ T cells, the cells comprising engineered human T cells containing multiple genetic modifications and anti-CD70 chimeric antigen receptor (CAR), wherein (a) the engineered human T cells contain genetic modifications within genomic coordinates chr6:29942891-29942915 of the HLA-A gene, genetic modifications within genomic coordinates chr6:31355222-31355246 of the HLA-B gene, genetic modifications within genomic coordinates chr16:10906643-10906667 of the CIITA gene, genetic modifications within genomic coordinates chr3:30674205-30674229 of the TGFBR2 gene, and genetic modifications within genomic coordinates chr19:6590121-6590145 of the CD70 gene, and (b) the anti-CD70... The CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region, the heavy chain variable region comprising a complementarity-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 75, and a light chain variable (VL) region, the light chain variable region comprising a complementarity-determining region 1 (VLCDR1) comprising the amino acid sequence of SEQ ID NO: 55, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted at genomic coordinates chr14:22547524-22547544 at the TRAC locus.

[0475] In some embodiments, in engineered human T cells, the VH region contains the amino acid sequence of SEQ ID NO: 45, and the VL region contains the amino acid sequence of SEQ ID NO: 39. In some embodiments, in engineered human T cells, the anti-CD70 CAR contains the amino acid sequence of SEQ ID NO: 4.

[0476] In some embodiments, the engineered human T cells are CD4+ or CD8+ T cells. In some embodiments, the engineered human T cells are CD4+ T cells. In some embodiments, the engineered human T cells are CD8+ T cells.

[0477] In some implementations, the engineered human T cells are homozygous for HLA-C. In some implementations, the engineered human T cells are homozygous for both HLA-B and HLA-C.

[0478] In some embodiments, this document provides a method for administering the above-described engineered human T cells or pharmaceutical composition to a subject in need. In some embodiments, this document provides a method for administering the above-described engineered human T cells or pharmaceutical composition to a subject as an adoptive cell transfer (ACT) therapy.

[0479] In some embodiments, this document provides a method for treating a disease or condition, the method comprising administering the aforementioned engineered human T cells or pharmaceutical composition to a subject in need.

[0480] In some embodiments, this document provides the engineered human T cells or pharmaceutical compositions described above for administration to a subject as an adoptive cell transfer (ACT) therapy. In some embodiments, this document provides the engineered human T cells or pharmaceutical compositions described above for treating a subject with cancer. In some embodiments, this document provides the engineered human T cells or pharmaceutical compositions described above for treating a subject with an infectious disease. In some embodiments, this document provides the engineered human T cells or pharmaceutical compositions described above for treating a subject with an autoimmune disease.

[0481] In some implementations, the disease or symptom is cancer. In some implementations, the cancer is a solid tumor or a hematologic malignancy. In some implementations, the solid tumor is renal cell carcinoma. In some implementations, the hematologic malignancy is acute myeloid leukemia. In some implementations, the hematologic malignancy is multiple myeloma.

[0482] Methods and compositions for generating cells containing anti-CD70 chimeric antigen receptor (CAR) and with reduced or eliminated surface expression of one or more of HLA-A, HLA-B, TRAC, MHC class II, TGFBR2, and CD70. In some embodiments, a method for manufacturing engineered cells is provided, the method comprising contacting the cells with: (a) a nucleic acid or mRNA encoding an anti-CD70 CAR, or an expression vector encoding a nucleic acid or mRNA encoding an anti-CD70 CAR; and (b) at least one genome editing tool comprising a genome editor and at least one guide RNA, wherein the at least one guide RNA targets a genomic locus selected from HLA-A, HLA-B, TRAC, CIITA, TGFBR2, and CD70 loci.

[0483] In some embodiments, a method for manufacturing engineered cells is provided, the method comprising (a) providing engineered cells having surface expression of one or both of TGFBR2 and CD70 reduced or eliminated relative to unmodified cells; and (b) contacting the cells with a nucleic acid or mRNA encoding an anti-CD70 CAR or an expression vector encoding a nucleic acid or mRNA (the nucleic acid or mRNA encoding an anti-CD70 CAR).

[0484] In some embodiments, a method for manufacturing engineered cells is provided, the method comprising (a) providing engineered cells having surface expression of one or more of HLA-A, HLA-B, MHC class II, TRAC, TGFBR2, and CD70 reduced or eliminated relative to unmodified cells; and (b) contacting the cells with a nucleic acid or mRNA encoding an anti-CD70 CAR or an expression vector encoding a nucleic acid or mRNA (the nucleic acid or mRNA encoding an anti-CD70 CAR).

[0485] 1. Anti-CD70 CAR knock-in This disclosure provides methods and compositions for generating cells comprising an anti-CD70 CAR encoded by a foreign nucleic acid. In some embodiments, the anti-CD70 CAR is the anti-CD70 CAR disclosed herein.

[0486] In some embodiments, the method includes contacting cells with a foreign nucleic acid encoding an anti-CD70 CAR. In some embodiments, the anti-CD70 CAR is the anti-CD70 CAR disclosed herein.

[0487] In some embodiments, a foreign nucleic acid encoding an anti-CD70 CAR is inserted into the genome of a target cell. In some embodiments, the foreign nucleic acid is integrated into the genome of the target cell. In some embodiments, the foreign nucleic acid is integrated into the genome of the target cell via homologous recombination (HR). In some embodiments, the foreign nucleic acid is integrated into the genome of the target cell via blunt-end insertion. In some embodiments, the foreign nucleic acid is integrated into the genome of the target cell via non-homologous end joining. In some embodiments, the foreign nucleic acid is integrated into a safe harbor locus in the cell genome. In some embodiments, the foreign nucleic acid is integrated into one of the TRAC locus or the CIITA locus. In some embodiments, the lipid-nucleic acid assembly composition is lipid nanoparticles (LNPs).

[0488] In some embodiments, the method produces a composition comprising engineered cells containing exogenous nucleic acids encoding anti-CD70 CAR.

[0489] In some embodiments, an allogeneic cell is provided, wherein the cell contains a foreign nucleic acid encoding anti-CD70CAR.

[0490] As used herein, the terms "exogenous nucleic acid," "template nucleic acid," or "donor template" refer to a nucleic acid to be inserted at or near a target sequence and which has been modified (e.g., cleaved) by the CRISPR system of this disclosure. In one embodiment, the endogenous nucleic acid sequence at or near the target site is modified to have some or all of the sequence of the exogenous nucleic acid, typically at or near one or more cleavage sites. In one embodiment, the exogenous nucleic acid is single-stranded. In an alternative embodiment, the exogenous nucleic acid is double-stranded. In one embodiment, the template nucleic acid is DNA, such as double-stranded DNA. In an alternative embodiment, the template nucleic acid is single-stranded DNA.

[0491] In some implementations, the exogenous nucleic acid contains a sequence encoding an anti-CD70 CAR (e.g., an anti-CD70 CAR as described herein).

[0492] In one implementation, the template nucleic acid alters the structure of the target site by participating in homology-directed repair events. In another implementation, the template nucleic acid alters the sequence of the target site. In yet another implementation, the template nucleic acid causes the incorporation of modified or non-naturally occurring bases into the target nucleic acid.

[0493] In one implementation, a single cleavage can be used to induce HDR. This document anticipates that a single cleavage can be used to increase the ratio of HDR, HR, or NHEJ at a given cleavage site.

[0494] A double-strand break or single-strand break in one strand should be close enough to the target site to allow correction to occur. In one embodiment, the distance is no more than 50, 100, 200, 300, 350, or 400 nucleotides. While not wishing to be bound by theory, it is believed that the break should be close enough to the target site that it lies within the region undergoing exonuclease-mediated removal during end excision. If the distance between the target site and the break is too large, the mutation may not be included in the end excision and may therefore be uncorrected, since the donor sequence can only be used to correct sequences within the end excision region.

[0495] The homologous arm should extend at least to the region where end resection can occur, for example, to allow the resected single strand to hang over a complementary region within the donor template. The total length may be limited by parameters such as plasmid size or viral packaging limitations. In one embodiment, the homologous arm does not extend into repeating elements, such as ALU repeats or LINE repeats. The template may have two homologous arms of the same or different lengths.

[0496] The illustrative homology arm length includes at least 25, 50, 100, 250, 500, 750, or 1000 nucleotides.

[0497] As used herein, a target site refers to a site on a target nucleic acid (e.g., a chromosome) that is modified by a Cas9 molecule-dependent process. For example, a target site can be a Cas9 molecule cleavage of the target nucleic acid and a targeted modification of the template nucleic acid at the target site, such as correction. In one embodiment, a target site can be a site between two nucleotides (e.g., adjacent nucleotides) on a target nucleic acid in which one or more nucleotides are added. A target site can contain one or more nucleotides that have been altered (e.g., corrected) by the template nucleic acid. In one embodiment, the target site is within a target sequence (e.g., a gRNA-binding sequence). In one embodiment, the target site is upstream or downstream of a target sequence (e.g., a gRNA-binding sequence).

[0498] Typically, the template sequence and the target sequence undergo break-mediated or catalytic recombination. In one embodiment, the template nucleic acid includes a sequence corresponding to a site on the target sequence that is cleaved by a Cas9-mediated cleavage event. In one embodiment, the template nucleic acid includes sequences corresponding to a first site on the target sequence that is cleaved in a first Cas9-mediated event and a second site on the target sequence that is cleaved in a second Cas9-mediated event.

[0499] The template nucleic acid comprises the following components: [5' homologous arm] - [insertion sequence] - [3' homologous arm]. The homologous arm provides for recombination into the chromosome, which can replace unwanted elements, such as mutations or imprints, with a substitution sequence. In one embodiment, the homologous arm is flanked by the most distal cleavage site.

[0500] In one embodiment, the 3' end of the 5' homologous arm is located immediately adjacent to the 5' end of the substitution sequence. In one embodiment, the 5' homologous arm may extend from the 5' end of the substitution sequence by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides.

[0501] In one embodiment, the 5' end of the 3' homologous arm is located immediately adjacent to the 3' end of the substitution sequence. In one embodiment, the 3' homologous arm may extend from the 3' end of the substitution sequence by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides.

[0502] This document anticipates that one or two homologous arms can be shortened to avoid including certain sequential repeating elements, such as Alu repeats or LINE elements. For example, the 5' homologous arm can be shortened to avoid sequential repeating elements. In other embodiments, the 3' homologous arm can be shortened to avoid sequential repeating elements. In some embodiments, both the 5' and 3' homologous arms can be shortened to avoid including certain sequential repeating elements.

[0503] This paper anticipates the design of template nucleic acids for mutation correction as single-stranded oligonucleotides (ssODNs). When using ssODNs, the lengths of the 5' and 3' homologous arms can range from approximately 200 base pairs (bp), for example, at least 25, 50, 75, 100, 125, 150, 175, or 200 bp. With continued improvements in oligonucleotide synthesis, longer homologous arms are also considered for ssODNs.

[0504] In one aspect, the insert sequence comprises a nucleic acid sequence encoding an anti-CD70 chimeric antigen receptor (e.g., as described herein). In one embodiment, the insert sequence further comprises a promoter, such as the EF-1α promoter, operatively linked to the nucleic acid sequence encoding the chimeric antigen receptor. In another aspect, the insert sequence comprises a vector encoding a chimeric antigen receptor (e.g., as described herein) or a portion thereof.

[0505] 2. HLA-A, HLA-B, TRAC, MHC Class II, TGFBR2, and CD70 knockout In some embodiments, the anti-CD70 CAR can be transduced into cells. In some embodiments, the method includes contacting the cells with donor nucleic acids encoding the anti-CD70 CAR for insertion into the cell genome.

[0506] In some embodiments, multiplex gene editing can be further performed on cells containing anti-CD70 CAR. In some embodiments, the method includes reducing or eliminating the surface expression of one or more of HLA-A, HLA-B, TRAC, or MHC class II proteins, which includes genetic modification of one or more of the HLA-A, HLA-B, TRAC, or CIITA genes, including contacting cells with a composition comprising one or more HLA-A, HLA-B, TRAC, or CIITA guide RNAs disclosed herein; and optionally an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0507] In some embodiments, the method includes reducing or eliminating the surface expression of one or more of the TGFBR2 or CD70 proteins, which includes genetically modifying one or more of the TGFBR2 or CD70 genes, including contacting cells with a composition comprising one or more TGFBR2 or CD70 guide RNAs disclosed herein; and optionally an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0508] In some embodiments, multiplex gene editing can be performed in cells. In some embodiments, the method includes reducing or eliminating the surface expression of HLA-A, HLA-B, TRAC, MHC class II, TGFBR2, and CD70 proteins and reducing or eliminating the expression of CIITA protein, which includes genetic modification of the HLA-A, HLA-B, TRAC, CIITA, TGFBR2, and CD70 genes, including contacting cells simultaneously with a composition comprising: a first composition comprising an HLA-A guide RNA disclosed herein; and optionally a first RNA-guided DNA binder or nucleic acid encoding an RNA-guided DNA binder; a second composition comprising an HLA-B guide RNA disclosed herein; and optionally a second RNA-guided DNA binder or nucleic acid encoding an RNA-guided DNA binder; and a third composition comprising a TRAC guide RNA disclosed herein; and optionally a third RNA-guided DNA binder. The fourth composition comprises a CIITA guide RNA disclosed herein, and optionally a fourth RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder; the fifth composition comprises a TGFBR2 guide RNA disclosed herein, and optionally a fifth RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder; the sixth composition comprises a CD70 guide RNA disclosed herein, and optionally a sixth RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder.

[0509] In some implementations, multiple gene editing can be performed sequentially. In other implementations, multiple gene editing can be performed sequentially.

[0510] In some implementation schemes, multiple gene editing can be performed simultaneously.

[0511] Multiplex gene editing can be performed using genome editors or genome editing components that are orthogonal to each other. As used herein, the term "orthogonal" refers to any two genome editors (e.g., base editors, nucleases, nickases, or lyases) that each can recognize one or more of its own targets via its homologous guide RNA, but is incompatible with one or more guide RNAs homologous to another genome editor; for example, neither can recognize one or more targets of the other genome editor via one or more guide RNAs homologous to the other genome editor. For instance, the Neisseria meningitidis Cas9 (NmeCas9) nickase may be able to recognize a genomic locus via a guide RNA homologous to the NmeCas9 nickase, and the Streptococcus pyogenes Cas9 (SpyCas9) lyase may be able to recognize another genomic locus via a guide RNA homologous to the SpyCas9 lyase. In this example, the NmeCas9 nickase and the SpyCas9 lyase are orthogonal to each other. Although in this example, the NmeCas9 nickase and SpyCas9 lyase are derived from different organisms, two genome editors do not need to be derived from different organisms to be orthogonal to each other.

[0512] In some embodiments, a method for genetically modifying cells is provided, the method comprising: (a) contacting the cells with a first genome editing tool, the first genome editing tool comprising a base editor and at least one guide RNA (gRNA) targeting at least one genomic locus and homologous to the base editor; and (b) contacting the cells with a second genome editing tool, the second genome editing tool comprising an RNA-guided lyase and at least one gRNA targeting at least one genomic locus and homologous to the RNA-guided lyase, wherein the base editor is orthogonal to the RNA-guided lyase, thereby generating at least two genome edits in the cells. In some embodiments, the method disclosed herein further comprises culturing cells to generate a cell population comprising edited cells, each of the edited cells containing at least two edits. In some embodiments, the base editor is a C-to-T base editor, optionally comprising a cytidine deaminase. In some embodiments, the RNA-guided DNA lyase comprises a *Streptococcus pyogenes* (Spy) Cas9 lyase and the base editor comprises a *Neisseria meningitidis* (Nme) Cas9 cleavage enzyme.

[0513] In other embodiments, the methods disclosed herein further include contacting cells with nucleic acids encoding an anti-CD70 CAR for insertion into an editing site (e.g., a double-strand break) at a TRAC locus. In other embodiments, the nucleic acid encoding the anti-CD70 CAR is contained within an expression vector. In other embodiments, the expression vector is an AAV vector.

[0514] In some embodiments, at least one gRNA homologous to a base editor targets one or more genes selected from the following loci: TGFBR2, CD70, HLA-A, HLA-B, and CIITA. In some embodiments, at least one gRNA homologous to an RNA-guided lyase targets the TRAC locus.

[0515] In some embodiments, at least one gRNA homologous to the base editor comprises one or more gRNAs selected from the following: gRNA targeting the HLA-A locus, gRNA targeting the HLA-B locus, gRNA targeting the CIITA locus, gRNA targeting the TGFBR2 locus, and gRNA targeting the CD70 locus, and at least one gRNA homologous to the RNA-guided lyase comprises a gRNA targeting the TRAC locus.

[0516] In some embodiments, at least one gRNA homologous to a base editor comprises a gRNA targeting the HLA-A locus and a gRNA targeting the CIITA locus, and at least one gRNA homologous to an RNA-guided lyase comprises a gRNA targeting the TRAC locus.

[0517] In some embodiments, at least one gRNA homologous to the base editor comprises one or more gRNAs selected from the following: gRNA targeting the HLA-A locus, gRNA targeting the HLA-B locus, and gRNA targeting the CIITA locus, and at least one gRNA homologous to the RNA-guided lyase comprises a gRNA targeting the TRAC locus.

[0518] In some embodiments, at least one gRNA homologous to the base editor comprises a gRNA targeting the TGFBR2 locus, one or more gRNAs selected from the following: a gRNA targeting the HLA-A locus, a gRNA targeting the HLA-B locus, a gRNA targeting the CIITA locus, and at least one gRNA homologous to the RNA-guided lyase comprises a gRNA targeting the TRAC locus.

[0519] In some embodiments, at least one gRNA homologous to the base editor comprises a gRNA targeting the CD70 locus and one or more gRNAs selected from the following: a gRNA targeting the HLA-A locus, a gRNA targeting the HLA-B locu...

Claims

1. An anti-CD70 chimeric antigen receptor (CAR), said anti-CD70 chimeric antigen receptor comprising: a. An antigen-binding protein or fragment thereof that specifically binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region including complementarity-determining region 1 (VH CDR1), VH CDR2, and VH CDR3, and a light chain variable (VL) region including complementarity-determining region 1 (VL CDR1), VL CDR2, and VL CDR3, and wherein... i. The VH CDR1 contains the amino acid sequence of any one of SEQ ID NO: 73, 67, 70, 76, 79 and 82; ii. The VH CDR2 comprises the amino acid sequence of any one of SEQ ID NO: 74, 68, 71, 77, 80, and 83; iii. The VH CDR3 comprises the amino acid sequence of any one of SEQ ID NO: 75, 69, 72, 78, 81, and 84; iv. The VL CDR1 comprises the amino acid sequence of any one of SEQ ID NO: 55, 49, 52, 58, 61 and 64; v. The VL CDR2 comprises the amino acid sequence of any one of SEQ ID NO: 56, 50, 53, 59, 62, and 65; and vi. The VL CDR3 comprises the amino acid sequence of any one of SEQ ID NO: 57, 51, 54, 60, 63 and 66; b. Transmembrane domains; and c. An intracellular domain comprising a co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 99 or 101.

2. The anti-CD70 CAR of claim 1, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the following amino acid sequence: (a) These are SEQ ID NOs: 73, 74, 75, 55, 56, and 57, respectively; (b) are SEQ ID NO: 67, 68, 69, 49, 50 and 51 respectively; (c) These are SEQ ID NOs: 70, 71, 72, 52, 53 and 54, respectively; (d) are SEQ ID NO: 76, 77, 78, 58, 59 and 60 respectively; (e) are SEQ ID NO: 79, 80, 81, 61, 62 and 63 respectively; or (f) are SEQ ID NO: 82, 83, 84, 64, 65 and 66 respectively.

3. The anti-CD70 CAR as described in claim 1 or 2, wherein the VH CDR1, the VH CDR2, the VHCDR3, the VL CDR1, the VL CDR2 and the VL CDR3 comprise the following amino acid sequences: SEQ ID NO: 73, 74, 75, 55, 56 and 57, respectively.

4. The anti-CD70 CAR according to any one of claims 1-3, wherein the VH region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO: 43-48.

5. The anti-CD70 CAR of any one of claims 1-4, wherein the VL region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 37-42.

6. The anti-CD70 CAR as described in any one of claims 1-5, wherein: (a) The VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 45, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 39; (b) The VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 43, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:

37. (c) The VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 44, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 38; (d) The VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 46, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

40. (e) The VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 47, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 41; or (f) The VH region contains an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 48, and the VL region contains an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

42.

7. The anti-CD70 CAR as described in any one of claims 1-6, wherein: The VH region contains an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 45, and the VL region contains an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

39.

8. The anti-CD70 CAR as described in any one of claims 1-7, wherein: (a) The VH region contains the amino acid sequence of SEQ ID NO: 45, and the VL region contains the amino acid sequence of SEQ ID NO: 39; (b) The VH region contains the amino acid sequence of SEQ ID NO: 43, and the VL region contains the amino acid sequence of SEQ ID NO: 37; (c) The VH region contains the amino acid sequence of SEQ ID NO: 44, and the VL region contains the amino acid sequence of SEQ ID NO: 38; (d) The VH region contains the amino acid sequence of SEQ ID NO: 46, and the VL region contains the amino acid sequence of SEQ ID NO: 40; (e) The VH region contains the amino acid sequence of SEQ ID NO: 47, and the VL region contains the amino acid sequence of SEQ ID NO: 41; or (f) The VH region contains the amino acid sequence of SEQ ID NO: 48, and the VL region contains the amino acid sequence of SEQ ID NO:

42.

9. The anti-CD70 CAR as described in any one of claims 1-8, wherein: The VH region contains the amino acid sequence of SEQ ID NO: 45, and the VL region contains the amino acid sequence of SEQ ID NO:

39.

10. The anti-CD70 CAR of any one of claims 1-9, wherein the antigen-binding protein comprises the amino acid sequence of any one of SEQ ID NO: 25, 27, 29, 32, 34 and 36, or a sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO: 25, 27, 29, 32, 34 and 36.

11. The anti-CD70 CAR of any one of claims 1-10, wherein the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25 or 27.

12. The anti-CD70 CAR of any one of claims 1-11, wherein the transmembrane domain comprises a CD8a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 95, or wherein the transmembrane domain comprises a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:

93.

13. The anti-CD70 CAR of any one of claims 1-12, wherein the anti-CD70 CAR further comprises a hinge domain between the antigen-binding protein and the transmembrane domain, optionally wherein the hinge domain is a CD8a hinge domain or a fragment thereof comprising the amino acid sequence of SEQ ID NO:

89.

14. The anti-CD70 CAR of any one of claims 1-13, wherein the intracellular domain further comprises an activation domain, optionally wherein the activation domain is a CD3z activation domain comprising the amino acid sequence of SEQ ID NO:

103.

15. The anti-CD70 CAR as described in any one of claims 1-11, wherein: a. The antigen-binding protein comprises the sequence of SEQ ID NO: 32 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; b. The antigen-binding protein comprises the sequence of SEQ ID NO: 32 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; c. The antigen-binding protein comprises the sequence of SEQ ID NO: 25 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; d. The antigen-binding protein comprises the sequence of SEQ ID NO: 25 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; e. The antigen-binding protein comprises the sequence of SEQ ID NO: 27 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; f. The antigen-binding protein comprises the sequence of SEQ ID NO: 27 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; g. The antigen-binding protein comprises the sequence of SEQ ID NO: 36 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; or h. The antigen-binding protein comprises the sequence of SEQ ID NO: 36 and the co-stimulatory domain comprises the sequence of SEQ ID NO:

99.

16. The anti-CD70 CAR according to any one of claims 1-15, wherein the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of any one of SEQ ID NO:2, 4, 6, 8, 14, 16, 20 and 22.

17. The anti-CD70 CAR according to any one of claims 1-16, wherein the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:2, 4 or 8.

18. The anti-CD70 CAR of any one of claims 1-17, wherein the antigen-binding protein is scFv.

19. The anti-CD70 CAR of claim 18, wherein the scFv comprises a linker between the VH region and the VL region, optionally wherein the scFv comprises a glycine-serine linker between the VH region and the VL region, and optionally the glycine-serine linker comprises the sequence of SEQ ID NO:

940.

20. A nucleic acid encoding an anti-CD70 CAR as described in any one of claims 1-19.

21. The nucleic acid of claim 20, wherein the nucleic acid comprises a nucleic acid sequence of any one of SEQ ID NO: 23, 24, 26, 28, 30, 31, 33 and 35, or a nucleic acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO: 23, 24, 26, 28, 30, 31, 33 and 35.

22. The nucleic acid of claim 20 or 21, wherein the nucleic acid comprises a nucleic acid sequence of any one of SEQ ID NO: 96-98 and 100, or a nucleic acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO: 96-98 and 100.

23. The nucleic acid of any one of claims 20-22, wherein the anti-CD70 CAR comprises a hinge domain, wherein the nucleic acid comprises a nucleic acid encoding the hinge domain, the hinge domain comprising a nucleic acid sequence of any one of SEQ ID NO: 85-88, or a nucleic acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 85-88.

24. The nucleic acid of any one of claims 20-23, wherein the nucleic acid comprises a nucleic acid sequence encoding the transmembrane domain, the transmembrane domain comprising a sequence of any one of SEQ ID NO: 90-92 and 94, or a sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 90-92 and 94.

25. The nucleic acid of any one of claims 20-24, wherein the intracellular domain comprises an activation domain, wherein the nucleic acid comprises a nucleic acid encoding the activation domain, and the activation domain comprises a nucleic acid sequence of SEQ ID NO: 102 or 104, or a nucleic acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 102 or 104.

26. The nucleic acid according to any one of claims 20-25, wherein the nucleic acid comprises a nucleic acid sequence of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21, or a nucleic acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21.

27. The nucleic acid according to any one of claims 20-26, wherein the nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NO: 1, 3 and 7.

28. An mRNA, said mRNA being encoded by a nucleic acid as described in any one of claims 20-27.

29. An expression vector operatively ligated to or comprising a nucleic acid as described in any one of claims 20-27.

30. An engineered cell comprising the nucleic acid as described in any one of claims 20-27, the mRNA as described in claim 28, or the expression vector as described in claim 29.

31. An engineered cell comprising an anti-CD70 CAR as claimed in any one of claims 1-19, optionally wherein the cell is transduced with an expression vector operatively linked to or comprising a nucleic acid encoding the anti-CD70 CAR, and optionally wherein the expression vector directs the expression of the anti-CD70 CAR in the cell.

32. The expression vector of claim 29 or the engineered cell of claim 30 or 31, wherein the expression vector comprises a retroviral or lentiviral expression vector.

33. The expression vector of claim 29 or the engineered cell of claim 30 or 31, wherein the expression vector comprises an AAV vector.

34. The expression vector or engineered cell of claim 33, wherein the expression vector comprises SEQ ID NO: 106, and optionally wherein the engineered cell comprises SEQ ID NO:

107.

35. An engineered cell comprising an anti-CD70 chimeric antigen receptor (CAR), wherein the anti-CD70 CAR comprises: a. An antigen-binding protein or fragment thereof that specifically binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region including complementarity-determining region 1 (VH CDR1), VH CDR2, and VH CDR3, and a light chain variable (VL) region including complementarity-determining region 1 (VL CDR1), VL CDR2, and VL CDR3, and wherein... i. The VH CDR1 contains the amino acid sequence of any one of SEQ ID NO: 73, 67, 70, 76, 79 and 82; ii. The VH CDR2 comprises the amino acid sequence of any one of SEQ ID NO: 74, 68, 71, 77, 80, and 83; iii. The VH CDR3 comprises the amino acid sequence of any one of SEQ ID NO: 75, 69, 72, 78, 81, and 84; iv. The VL CDR1 comprises the amino acid sequence of any one of SEQ ID NO: 55, 49, 52, 58, 61 and 64; v. The VL CDR2 comprises the amino acid sequence of any one of SEQ ID NO: 56, 50, 53, 59, 62 and 65; vi. The VL CDR3 comprises the amino acid sequence of any one of SEQ ID NO: 57, 51, 54, 60, 63 and 66; b. Transmembrane domains; and c. An intracellular domain comprising a co-stimulatory domain comprising the amino acid sequence of SEQ ID NO: 99 or 101.

36. The engineered cell of claim 35, wherein the VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the following amino acid sequence: (a) These are SEQ ID NOs: 73, 74, 75, 55, 56, and 57, respectively; (b) are SEQ ID NO: 67, 68, 69, 49, 50 and 51 respectively; (c) These are SEQ ID NOs: 70, 71, 72, 52, 53 and 54, respectively; (d) are SEQ ID NO: 76, 77, 78, 58, 59 and 60 respectively; (e) are SEQ ID NO: 79, 80, 81, 61, 62 and 63 respectively; or (f) are SEQ ID NO: 82, 83, 84, 64, 65 and 66 respectively.

37. The engineered cell of claim 35 or 36, wherein the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 comprise the following amino acid sequence: They are SEQ ID NO: 73, 74, 75, 55, 56 and 57 respectively.

38. The engineered cell according to any one of claims 35-37, wherein the VH region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 43-48.

39. The engineered cell according to any one of claims 35-38, wherein the VL region comprises an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 37-42.

40. The engineered cell according to any one of claims 35-39, wherein: (a) The VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 45, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 39; (b) The VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 43, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:

37. (c) The VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 44, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 38; (d) The VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 46, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

40. (e) The VH region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 47, and the VL region contains an amino acid sequence having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 41; or (f) The VH region contains an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 48, and the VL region contains an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

42.

41. The engineered cell according to any one of claims 35-40, wherein: The VH region contains an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 45, and the VL region contains an amino acid sequence that is at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

39.

42. The engineered cell according to any one of claims 35-41, wherein: (a) The VH region contains the amino acid sequence of SEQ ID NO: 45, and the VL region contains the amino acid sequence of SEQ ID NO: 39; (b) The VH region contains the amino acid sequence of SEQ ID NO: 43, and the VL region contains the amino acid sequence of SEQ ID NO: 37; (c) The VH region contains the amino acid sequence of SEQ ID NO: 44, and the VL region contains the amino acid sequence of SEQ ID NO: 38; (d) The VH region contains the amino acid sequence of SEQ ID NO: 46, and the VL region contains the amino acid sequence of SEQ ID NO: 40; (e) The VH region contains the amino acid sequence of SEQ ID NO: 47, and the VL region contains the amino acid sequence of SEQ ID NO: 41; or (f) The VH region contains the amino acid sequence of SEQ ID NO: 48, and the VL region contains the amino acid sequence of SEQ ID NO:

42.

43. The engineered cell according to any one of claims 35-42, wherein: The VH region contains the amino acid sequence of SEQ ID NO: 45, and the VL region contains the amino acid sequence of SEQ ID NO:

39.

44. The engineered cell according to any one of claims 35-43, wherein the antigen-binding protein comprises the amino acid sequence of any one of SEQ ID NO: 25, 27, 29, 32, 34 and 36, or a sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO: 25, 27, 29, 32, 34 and 36.

45. The engineered cell according to any one of claims 35-44, wherein the antigen-binding protein comprises the amino acid sequence of SEQ ID NO: 25 or 27.

46. ​​The engineered cell according to any one of claims 35-45, wherein the transmembrane domain comprises a CD8a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 95, or wherein the transmembrane domain comprises a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO:

93.

47. The engineered cell according to any one of claims 35-46, wherein the engineered cell further comprises a hinge domain between the antigen-binding protein and the transmembrane domain, optionally wherein the hinge domain is a CD8a hinge domain or a fragment thereof comprising the amino acid sequence of SEQ ID NO:

89.

48. The engineered cell according to any one of claims 35-47, wherein the intracellular domain further comprises an activation domain, optionally wherein the activation domain is a CD3z activation domain comprising the amino acid sequence of SEQ ID NO:

103.

49. The engineered cell according to any one of claims 35-48, wherein: a. The antigen-binding protein comprises the sequence of SEQ ID NO: 32 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; b. The antigen-binding protein comprises the sequence of SEQ ID NO: 32 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; c. The antigen-binding protein comprises the sequence of SEQ ID NO: 25 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; d. The antigen-binding protein comprises the sequence of SEQ ID NO: 25 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; e. The antigen-binding protein comprises the sequence of SEQ ID NO: 27 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; f. The antigen-binding protein comprises the sequence of SEQ ID NO: 27 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 99; g. The antigen-binding protein comprises the sequence of SEQ ID NO: 36 and the co-stimulatory domain comprises the sequence of SEQ ID NO: 101; or h. The antigen-binding protein comprises the sequence of SEQ ID NO: 36 and the co-stimulatory domain comprises the sequence of SEQ ID NO:

99.

50. The engineered cell according to any one of claims 35-49, wherein the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequences of SEQ ID NO: 2, 4, 6, 8, 14, 16, 20 and 22.

51. The engineered cell according to any one of claims 35-50, wherein the anti-CD70 CAR comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 2, 4 or 8.

52. The engineered cell according to any one of claims 35-51, wherein the antigen-binding protein is scFv.

53. The engineered cell of claim 52, wherein the scFv comprises a linker between the VH region and the VL region, optionally wherein the scFv comprises a glycine-serine linker between the VH region and the VL region, optionally the linker comprises the sequence of SEQ ID NO:

940.

54. A cell population, wherein the cell population comprises engineered cells as claimed in any one of claims 35-53.

55. The engineered cells or cell populations according to any one of claims 35-54, wherein the engineered cells or cell populations further comprise reduced or eliminated TGFBR2 surface expression relative to unmodified cells.

56. The engineered cell or cell population according to any one of claims 35-55, wherein the engineered cell or cell population further comprises a gene modification in the TGFBR2 gene, optionally wherein the gene modification comprises at least one nucleotide in the following genomic coordinates: chr3:30606864-30691614.

57. The engineered cell or cell population of claim 56, wherein the gene modification is located within genomic coordinates chr3:30674205-30674229, optionally wherein the gene modification comprises at least one nucleotide within genomic coordinates targeted by a TGFBR2 guide RNA containing the guide sequence of SEQ ID NO:

301.

58. The engineered cell or cell population of claim 56, wherein the gene modification is located within genomic coordinates chr3:30671941-30671961, optionally wherein the gene modification comprises at least one nucleotide within genomic coordinates targeted by a TGFBR2 guide RNA containing the guide sequence of SEQ ID NO:

302.

59. The engineered cells or cell populations according to any one of claims 35-58, wherein the engineered cells or cell populations further comprise reduced or eliminated CD70 surface expression relative to unmodified cells.

60. The engineered cell or cell population according to any one of claims 35-59, wherein the engineered cell or cell population further comprises a gene modification in the CD70 gene, optionally wherein the gene modification comprises at least one nucleotide within genomic coordinates chr19:6586002-6591018, or optionally wherein the gene modification is within genomic coordinates chr19:6586028-6591018.

61. The engineered cell or cell population of claim 60, wherein the gene modification is located within the following genomic coordinates: chr19:6590121-6590145 or chr19:6586268-6586292, optionally wherein the gene modification comprises at least one nucleotide within the genomic coordinates targeted by a CD70 guide RNA containing the guide sequence of SEQ ID NO: 310 or 311.

62. The engineered cell or cell population of claim 60, wherein the gene modification comprises at least one nucleotide in the following genomic coordinates: chr19:6590998-6591018 or chr19:6590991-6591011, optionally wherein the gene modification comprises at least one nucleotide in the genomic coordinates targeted by a CD70 guide RNA comprising the guide sequence of SEQ ID NO: 312 or 313.

63. The engineered cells or cell populations as described in any one of claims 35-62, wherein the engineered cells or cell populations further comprise reduced or eliminated HLA-A surface expression relative to unmodified cells.

64. The engineered cell or cell population according to any one of claims 35-63, wherein the engineered cell or cell population further comprises a gene modification in the HLA-A gene, optionally wherein the gene modification comprises at least one nucleotide in the following genomic coordinates: (a) chr6:29942854-29942913 and chr6:29943518-29943619; and (b) chr6:29942540-29945459.

65. The engineered cell or cell population of claim 64, wherein the gene modification is within genomic coordinates selected from the following: chr6:29942891-29942915; and chr6:29942609-29942633, and optionally wherein the gene modification comprises at least one nucleotide within genomic coordinates targeted by an HLA-A guide RNA containing the guide sequence of SEQ ID NO: 403 or 404.

66. The engineered cells or cell populations according to any one of claims 35-65, wherein the engineered cells or cell populations have reduced or eliminated HLA-B surface expression relative to unmodified cells.

67. The engineered cell or cell population according to any one of claims 35-66, wherein the engineered cell or cell population further comprises a gene modification in the HLA-B gene, optionally wherein the gene modification is located in genomic coordinates selected from the following: (a) chr6:31354480-31357174 and (b) chr6:31357084-31354647.

68. The engineered cell of claim 67, wherein the gene modification is within genomic coordinates selected from the following: chr6:31355222-31355246, chr6:31355221-31355245 and chr6:31355205-31355229, optionally wherein the gene modification comprises at least one nucleotide within genomic coordinates targeted by an HLA-B guide RNA containing a guide sequence of SEQ ID NO: 406, 405 or 407.

69. The engineered cell or cell population according to any one of claims 35-68, wherein the engineered cell or cell population comprises reduced or eliminated TRAC surface expression relative to unmodified cells.

70. The engineered cell or cell population as described in claims 35-69, wherein the engineered cell or cell population comprises the gene modification in the TRAC gene.

71. The engineered cell or cell population of claim 70, wherein the gene modification comprises at least one nucleotide within genomic coordinates chr14:22547524-22547544, optionally wherein the gene modification comprises at least one nucleotide within genomic coordinates targeted by a TRAC guide RNA comprising the guide sequence of SEQ ID NO:

413.

72. The engineered cells or cell populations according to any one of claims 35-71, wherein the engineered cells or cell populations have reduced or eliminated surface expression of MHC class II relative to unmodified cells.

73. The engineered cell or cell population according to any one of claims 35-72, wherein the engineered cell or cell population further comprises a gene modification in the CIITA gene, optionally wherein the gene modification is within the gene coordinates selected from the following: (a) chr16:10877363-10907788 and (b) chr16:10906515-10908136.

74. The engineered cell or cell population of claim 73, wherein the gene modification comprises at least one nucleotide within genomic coordinates chr16:10906643-10906667 or chr16:10907504-10907528, optionally wherein the gene modification comprises at least one nucleotide within genomic coordinates targeted by a CIITA guide RNA comprising the guide sequence of SEQ ID NO: 402 or 401.

75. The engineered cell or cell population according to any one of claims 56 to 74, wherein the gene modification comprises insertion / deletion, C to T substitution, or A to G substitution within the genomic coordinates.

76. An engineered cell comprising a gene modification in the HLA-A gene, a modified TRAC gene, a gene modification in the CIITA gene, a gene modification in the TGFBR2 gene, and / or a gene modification in the CD70 gene, wherein the engineered cell expresses an anti-CD70 chimeric antigen receptor (CAR) or contains nucleic acid encoding an anti-CD70 CAR.

77. An engineered cell comprising gene modifications in the HLA-A gene, the HLA-B gene, the TRAC gene, the CIITA gene, the TGFBR2 gene, and / or the CD70 gene, wherein the engineered cell expresses an anti-CD70 chimeric antigen receptor (CAR) or contains nucleic acid encoding an anti-CD70 CAR.

78. The engineered cell or cell population according to any one of claims 35-77, wherein the cell is homozygous for HLA-C, and optionally wherein the cell is homozygous for both HLA-B and HLA-C.

79. A pharmaceutical composition comprising engineered cells or cell populations as described in any one of claims 35-78.

80. The engineered cell, cell population, or pharmaceutical composition according to any one of claims 35-79, wherein the genetic modification comprises the insertion of a heterologous coding sequence.

81. The engineered cell, cell population, or pharmaceutical composition according to any one of claims 35-80, wherein the engineered cell is an immune cell.

82. The engineered cell, cell population, or pharmaceutical composition according to any one of claims 35-81, wherein the cell is an NK cell.

83. The engineered cell, cell population, or pharmaceutical composition according to any one of claims 35-81, wherein the cell is a T cell, optionally wherein the T cell is a CD4+ T cell, optionally wherein the T cell is a CD8+ T cell, or optionally wherein the T cell has a T memory stem cell (Tscm) phenotype.

84. The engineered cell, cell population, or pharmaceutical composition according to any one of claims 35-83, wherein the cell is engineered using a genome editing system.

85. The engineered cell, cell population, or pharmaceutical composition of claim 84, wherein the genome editing system comprises an RNA-guided DNA binder or a nucleic acid encoding an RNA-guided DNA binder, optionally wherein the RNA-guided DNA binder or the RNA-guided DNA binder encoded by the nucleic acid is Streptococcus pyogenes Cas9 (SpyCas9), or optionally wherein the RNA-guided DNA binder or the RNA-guided DNA binder encoded by the nucleic acid is Neisseria meningitidis Cas9 (NmeCas9).

86. The engineered cell, cell population, or pharmaceutical composition according to any one of claims 57-85, wherein the guide RNA is provided to the cell in a vector, and / or wherein the RNA-guided DNA binder is provided to the cell in a vector, optionally in the same vector as the guide RNA.

87. The engineered cell, cell population, or pharmaceutical composition according to any one of claims 35-86, wherein the nucleic acid encoding the anti-CD70 CAR is provided to the cell in an expression vector.

88. The engineered cell, cell population, or pharmaceutical composition of claim 87, wherein the expression vector is a viral vector, optionally wherein the expression vector comprises an AAV vector, and optionally wherein the expression vector comprises SEQ ID NO: 106 or 107.

89. The engineered cell, cell population, or pharmaceutical composition of claim 87, wherein the expression vector is a non-viral vector.

90. The engineered cell, cell population, or pharmaceutical composition according to any one of claims 57-89, wherein the guide RNA is provided to the cell in a lipid nanoparticle (LNP), optionally in the same LNP providing an RNA-guided DNA binder.

91. The engineered cell, cell population, or pharmaceutical composition according to any one of claims 35-90, wherein the nucleic acid encoding the anti-CD70 CAR is provided to the cell in lipid nanoparticles (LNPs).

92. A method for manufacturing engineered cells, the method comprising contacting the cells with: a. The nucleic acid as described in any one of claims 20-27, or the mRNA as described in claim 28, or the expression vector as described in claim 29; and b. At least one genome editing tool comprising a genome editor and at least one guide RNA, wherein the at least one guide RNA targets a genomic locus selected from HLA-A, HLA-B, TRAC, CIITA, TGFBR2, and CD70 loci.

93. A method for manufacturing engineered cells comprising anti-CD70 CAR, the method comprising: a. Providing engineered cells, said engineered cells having reduced or eliminated surface expression of one or both of TGFBR2 and CD70 relative to unmodified cells; and b. Contact the cells with the nucleic acid as described in any one of claims 20-27, or the mRNA as described in claim 28, or the expression vector as described in claim 29.

94. A method for manufacturing engineered cells comprising anti-CD70 CAR, the method comprising: a. Providing engineered cells having reduced or eliminated surface expression of one or more of HLA-A, HLA-B, MHC class II, TRAC, TGFBR2, and CD70 compared to unmodified cells; and b. Contact the cells with the nucleic acid as described in any one of claims 20-27, or the mRNA as described in claim 28, or the expression vector as described in claim 29.

95. A method for manufacturing engineered cells comprising anti-CD70 CAR, the method comprising: (a) Contacting the cells with a first set of lipid nanoparticles (LNPs), the first set of LNPs comprising an LNP containing a UGI and at least one LNP containing a base editor and at least one guide RNA, the at least one guide RNA being homologous to the base editor and targeting a genomic locus selected from HLA-A, HLA-B, TRAC, CIITA, TGFBR2, and CD70 loci; and (b) Expose the cells to the following: i. A second set of LNPs, comprising an LNP containing a UGI and at least one LNP containing a base editor and at least one guide RNA, wherein the at least one guide RNA is homologous to the base editor and targets a genomic locus selected from HLA-A, HLA-B, TRAC, CIITA, TGFBR2, and CD70 loci, wherein the at least one guide RNA is different from the at least one guide RNA contained in the first set of LNPs of step (a); and ii. At least one LNP comprising an RNA-guided lyase and at least one gRNA, wherein the at least one gRNA is homologous to the RNA-guided lyase and targets the TRAC locus; and iii. Nucleic acid encoding anti-CD70 CAR for insertion into an editing site (e.g., a double-strand break) at the TRAC locus.

96. A method for manufacturing engineered cells comprising anti-CD70 CAR, the method comprising: (a) Contacting cells with: a first group of lipid nanoparticles (LNPs) comprising a first LNP containing a base editor and gRNA targeting the HLA-A locus; a second LNP containing a base editor and gRNA targeting the HLA-B locus; a third LNP containing a base editor and gRNA targeting the CIITA locus; and a fourth LNP containing a uracil glycosidase inhibitor (UGI); (b) Contacting cells with: (i) a second group of LNPs, the second group of LNPs containing a base editor and gRNA targeting the CIITA locus; and a fourth LNP containing a uracil glycosidase inhibitor (UGI); The LNP comprises a fifth LNP containing a base editor and a gRNA targeting the TGFBR2 locus; a sixth LNP containing a base editor and a gRNA targeting the CD70 locus; (ii) a seventh LNP containing an RNA-guided DNA lyase and a gRNA homologous to the RNA-guided DNA lyase and targeting the TRAC locus; an eighth lipid LNP containing a UGI; and (iii) a nucleic acid encoding an anti-CD70 CAR for insertion into an editing site (e.g., a double-strand break) at the TRAC locus.

97. The method of claim 95 or 96, wherein the RNA-guided lyase comprises a Streptococcus pyogenes (Spy) Cas9 lyase, and the base editor comprises a Neisseria meningitidis (Nme) Cas9 nickase.

98. A method of administering, to a subject in need or as an adoptive cell transfer (ACT) therapy, the engineered cells, cell populations or pharmaceutical composition as described in any one of claims 35-91.

99. A method of treating a disease or condition, the method comprising administering to a subject in need an engineered cell, cell population, or pharmaceutical composition as described in any one of claims 35-91.

100. The method of claim 98 or 99, wherein the engineered cells are allogeneic to the subject.

101. The engineered cells, cell populations, compositions, or methods according to any one of claims 35-91, for administration to a subject as an adoptive cell transfer (ACT) therapy, for treating a subject with cancer, for treating a subject with an infectious disease, or for treating a subject with an autoimmune disease.

102. Use of an engineered cell, cell population, or pharmaceutical composition as described in any one of claims 35-91 for the manufacture of a medicament for treating a subject suffering from cancer, an infectious disease, or an autoimmune disease.

103. An engineered cell comprising gene modifications in the HLA-A gene, the HLA-B gene, the TRAC gene, the CIITA gene, the TGFBR2 gene, and / or the CD70 gene, wherein the gene modification in the HLA-A gene is located within genomic coordinates chr6:29942891-29942915; wherein the gene modification in the HLA-B gene is located within genomic coordinates chr6:31355222-31355246, chr6:31355221-31355245, or chr6:31355205-31355229; and wherein the gene modification in the TRAC gene is located within genomic coordinates chr14: Within 22547524-22547544; wherein the gene modification in the CIITA gene is within genomic coordinates chr16:10907504-10907528; wherein the gene modification in the TGFBR2 gene is within genomic coordinates chr3:30674205-30674229 or chr3:30671941-30671961; and wherein the gene modification in the CD70 gene is within genomic coordinates chr19:6590121-6590145 or chr19:6590998-6591018, wherein the engineered cells contain anti-CD70 chimeric antigen receptor (CAR) or contain nucleic acid encoding anti-CD70 CAR.

104. An engineered cell comprising gene modifications in the HLA-A gene, the HLA-B gene, the TRAC gene, the CIITA gene, the TGFBR2 gene, and / or the CD70 gene, wherein the gene modification in the HLA-A gene is located within genomic coordinates chr6:29942891-29942915; wherein the gene modification in the HLA-B gene is located within genomic coordinates chr6:31355222-31355246; wherein the gene modification in the TRAC gene... The modifications are located within genomic coordinates chr14:22547524-22547544; the gene modifications in the CIITA gene are located within genomic coordinates chr16:10906643-10906667; the gene modifications in the TGFBR2 gene are located within genomic coordinates chr3:30674205-30674229; and the gene modifications in the CD70 gene are located within genomic coordinates chr19:6590121-6590145, wherein the engineered cells contain anti-CD70 chimeric antigen receptor (CAR) or contain nucleic acids encoding anti-CD70 CAR.

105. An engineered human T cell comprising multiple genetic modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein... (a) The engineered human T cells comprise HLA-A gene modifications and reduced or eliminated HLA-A surface expression relative to unmodified cells, HLA-B gene modifications and reduced or eliminated HLA-B surface expression relative to unmodified cells, CIITA gene modifications and reduced or eliminated MHC class II surface expression relative to unmodified cells, TGFBR2 gene modifications and reduced or eliminated TGFBR2 surface expression relative to unmodified cells, and CD70 gene modifications and reduced or eliminated CD70 surface expression relative to unmodified cells. (b) The anti-CD70 CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region comprising a complementation-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 75, and a light chain variable (VL) region comprising a complementation-determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 55, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted into the TRAC locus.

106. An engineered human T cell comprising multiple genetic modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein... (a) The engineered human T cells contain gene modifications within the genomic coordinates chr6:29942891-29942915 of the HLA-A gene, gene modifications within the genomic coordinates chr6:31355222-31355246 of the HLA-B gene, gene modifications within the genomic coordinates chr16:10906643-10906667 of the CIITA gene, gene modifications within the genomic coordinates chr3:30674205-30674229 of the TGFBR2 gene, and gene modifications within the genomic coordinates chr19:6590121-6590145 of the CD70 gene, and (b) The anti-CD70 CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region comprising a complementation-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 75, and a light chain variable (VL) region comprising a complementation-determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 55, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted into the TRAC locus.

107. An engineered human T cell comprising multiple genetic modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein... (a) The engineered human T cells contain gene modifications within the genomic coordinates chr6:29942891-29942915 of the HLA-A gene, gene modifications within the genomic coordinates chr6:31355222-31355246 of the HLA-B gene, gene modifications within the genomic coordinates chr16:10906643-10906667 of the CIITA gene, gene modifications within the genomic coordinates chr3:30674205-30674229 of the TGFBR2 gene, and gene modifications within the genomic coordinates chr19:6590121-6590145 of the CD70 gene, and (b) The anti-CD70 CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region comprising a complementation-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 75, and a light chain variable (VL) region comprising a complementation-determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 55, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted at genomic coordinates chr14:22547524-22547544 at the TRAC locus.

108. The engineered human T cell according to any one of claims 105-107, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 45, and the VL region comprises the amino acid sequence of SEQ ID NO:

39.

109. The engineered human T cell according to any one of claims 105-108, wherein the anti-CD70CAR comprises the amino acid sequence of SEQ ID NO:

4.

110. The engineered human T cell according to any one of claims 105-109, wherein the engineered human T cell is a CD4+ or CD8+ T cell.

111. The engineered human T cell according to any one of claims 105-110, wherein the engineered human T cell is homozygous for HLA-C, and optionally wherein the engineered human T cell is homozygous for both HLA-B and HLA-C.

112. A pharmaceutical composition comprising a T cell population, wherein the T cell population comprises CD4+ T cells and / or CD8+ T cells, said cells comprising engineered human T cells containing multiple genetic modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein (a) The engineered human T cells comprise HLA-A gene modifications and reduced or eliminated HLA-A surface expression relative to unmodified cells, HLA-B gene modifications and reduced or eliminated HLA-B surface expression relative to unmodified cells, CIITA gene modifications and reduced or eliminated MHC class II surface expression relative to unmodified cells, TGFBR2 gene modifications and reduced or eliminated TGFBR2 surface expression relative to unmodified cells, and CD70 gene modifications and reduced or eliminated CD70 surface expression relative to unmodified cells. (b) The anti-CD70 CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region comprising a complementation-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 75, and a light chain variable (VL) region comprising a complementation-determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 55, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted into the TRAC locus.

113. A pharmaceutical composition comprising a T cell population, wherein the T cell population comprises CD4+ T cells and / or CD8+ T cells, said cells comprising engineered human T cells containing multiple genetic modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein (a) The engineered human T cells contain gene modifications within the genomic coordinates chr6:29942891-29942915 of the HLA-A gene, gene modifications within the genomic coordinates chr6:31355222-31355246 of the HLA-B gene, gene modifications within the genomic coordinates chr16:10906643-10906667 of the CIITA gene, gene modifications within the genomic coordinates chr3:30674205-30674229 of the TGFBR2 gene, and gene modifications within the genomic coordinates chr19:6590121-6590145 of the CD70 gene, and (b) The anti-CD70 CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region comprising a complementation-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 75, and a light chain variable (VL) region comprising a complementation-determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 55, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted into the TRAC locus.

114. A pharmaceutical composition comprising a T cell population, wherein the T cell population comprises CD4+ T cells and / or CD8+ T cells, said cells comprising engineered human T cells containing multiple genetic modifications and an anti-CD70 chimeric antigen receptor (CAR), wherein (a) The engineered human T cells contain gene modifications within the genomic coordinates chr6:29942891-29942915 of the HLA-A gene, gene modifications within the genomic coordinates chr6:31355222-31355246 of the HLA-B gene, gene modifications within the genomic coordinates chr16:10906643-10906667 of the CIITA gene, gene modifications within the genomic coordinates chr3:30674205-30674229 of the TGFBR2 gene, and gene modifications within the genomic coordinates chr19:6590121-6590145 of the CD70 gene, and (b) The anti-CD70 CAR comprises an antigen-binding protein or a fragment thereof that binds to CD70, wherein the antigen-binding protein comprises a heavy chain variable (VH) region comprising a complementation-determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 73, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 75, and a light chain variable (VL) region comprising a complementation-determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 55, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 57, wherein the anti-CD70 CAR is inserted at genomic coordinates chr14:22547524-22547544 at the TRAC locus.

115. The pharmaceutical composition of any one of claims 112-114, wherein the VH region comprises the amino acid sequence of SEQ ID NO:45, and the VL region comprises the amino acid sequence of SEQ ID NO:

39.

116. The pharmaceutical composition of any one of claims 112-115, wherein the anti-CD70 CAR comprises the amino acid sequence of SEQ ID NO:

4.

117. The pharmaceutical composition of any one of claims 112-116, wherein the engineered human T cells are homozygous for HLA-C, and optionally wherein the engineered human T cells are homozygous for both HLA-B and HLA-C.

118. A method of administering engineered human T cells or a pharmaceutical composition as described in any one of claims 105-117 to a subject in need or as an adoptive cell transfer (ACT) therapy.

119. A method of treating a disease or condition, the method comprising administering to a subject in need an engineered human T cell or pharmaceutical composition as described in any one of claims 105-117.

120. The engineered human T cells or pharmaceutical composition of any one of claims 105-117, for administration to a subject as an adoptive cell transfer (ACT) therapy, for treating a subject with cancer, for treating a subject with an infectious disease, or for treating a subject with an autoimmune disease.

121. The method of claim 119, wherein the disease or condition is cancer.

122. The engineered human T cells or pharmaceutical composition for use as claimed in claim 120 or the method as claimed in claim 121, wherein the cancer is a solid tumor or a hematologic malignancy.

123. The engineered human T cells or pharmaceutical composition for use as claimed in claim 122, or the method as claimed in claim 122, wherein the solid tumor is renal cell carcinoma, or wherein the hematologic malignancy is acute myeloid leukemia or multiple myeloma.

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