Proteins and uses thereof

Abstract

The present application provides Nef proteins and Nef fusion proteins and methods of use thereof. The present application also provides engineered cells (e.g., CAR-T cells) having reduced levels of endogenous TCR, in particular engineered cells that overexpress a Nef protein or a Nef fusion protein.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to International Patent Application No. PCT / CN2023 / 106325, filed on July 7, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] The sequence list is submitted as an XML file.

[0004] The following content, submitted as an XML file, is incorporated herein by reference in its entirety: Sequence List in Computer-Readable Form (CRF) (filename: IEC240352PCT SEQUENCE LISTING.XML, record date: July 4, 2024, size: 38,569 KB). Technical Field

[0005] This disclosure relates to proteins used to downregulate targets and their uses. This disclosure also relates to engineered cells (e.g., CAR-T cells) that express these proteins. Background Technology

[0006] Chimeric antigen receptor (CAR)-T cell therapy utilizes genetically modified T cells carrying engineered receptors that specifically recognize target tumor antigens to guide T cells to the tumor site. It has shown promising results in treating hematologic malignancies and multiple myeloma (MM). However, autologous CAR-T or TCR-T therapies (using the patient's own T cells) present significant challenges in manufacturing and standardization due to individual variability, resulting in extremely high manufacturing and treatment costs. Furthermore, cancer patients often have weakened immune function, with reduced lymphocyte counts and immune activity, making in vitro expansion difficult.

[0007] Universal allogeneic CAR-T or TCR-T therapy is considered an ideal model, where T cells are derived from healthy donors. However, a key challenge is how to effectively eliminate graft-versus-host disease (GvHD), which is attributed to tissue incompatibility, during treatment. TCRs are cell surface receptors involved in T cell activation in response to antigen presentation. 95% of human T cells possess a TCR composed of α (alpha) and β (beta) chains. The TCR α and β chains combine to form a heterodimer and associate with the CD3 subunit to form the TCR complex present on the cell surface. GvHD occurs when donor T cells recognize non-self major histocompatibility complex (MHC) molecules via the TCR and perceive host (transplant recipient) tissue as foreign antigens, attacking them. To eliminate endogenous TCRs from donor T cells and thus prevent GvHD, gene editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regular-interval short palindromic repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR / Cas) have been used to knock out endogenous TCRα or TCRβ genes (KO), and then enrich TCR-negative T cells to generate allogeneic CAR-T or TCR-T.

[0008] Nef (negative regulator) is a small 27-35 kDa myristylated protein encoded by primate lentiviruses. Nef (e.g., SIV Nef) can mediate the downregulation of endogenous T cell receptors (TCRs) and can suppress inflammation. Summary of the Invention

[0009] This disclosure relates to Nef proteins and Nef fusion proteins, as well as methods of their preparation and use. This disclosure also relates to engineered cells with reduced levels of endogenous TCR, particularly engineered cells overexpressing Nef proteins (e.g., truncated Nef proteins) or Nef fusion proteins (e.g., Nef proteins fused with TRIM21 proteins). Compared to GvHD responses induced by primary T cells isolated from the donor of precursor T cells from which the engineered cells are derived, engineered cells may not induce a graft-versus-host disease (GvHD) response or induce a reduced GvHD response in tissue-incompatible individuals.

[0010] In one respect, this disclosure relates to a non-naturally occurring negative regulatory factor (Nef) protein containing amino acids 207-223 or the corresponding amino acids numbered in SEQ ID NO:1 and SEQ ID NO:2.

[0011] In some embodiments, the non-naturally occurring Nef protein comprises the deletion of amino acids 50-91, as numbered in SEQ ID NO: 1 and SEQ ID NO: 2, or the corresponding amino acids thereof.

[0012] In some embodiments, the non-naturally occurring Nef protein downregulates the cell surface expression of the endogenous T cell receptor (TCR).

[0013] In some embodiments, the non-naturally occurring Nef protein consists of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

[0014] In one respect, this disclosure relates to a fusion protein comprising (1) a non-naturally occurring Nef protein and (2) an E3 ubiquitin ligase or a fragment thereof.

[0015] In some embodiments, the non-naturally occurring Nef protein comprises the deletion of amino acids 207-223 or the corresponding amino acids numbered in SEQ ID NO: 1 and SEQ ID NO: 2.

[0016] In some embodiments, the non-naturally occurring Nef protein comprises the deletion of amino acids 50-91, as numbered in SEQ ID NO: 1 and SEQ ID NO: 2, or the corresponding amino acids thereof.

[0017] In some embodiments, the non-naturally occurring Nef protein consists of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 or an amino acid sequence that is at least 90%, 95% or 99% identical to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0018] In some embodiments, E3 ubiquitin ligase or a fragment thereof is derived from the TripartiteMotif Containing 21 (TRIM21) protein.

[0019] In some embodiments, the TRIM21 protein is truncated.

[0020] In some embodiments, the TRIM21 protein comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12 or an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0021] In some embodiments, the amino acid sequence of the TRIM21 protein is shown in SEQ ID NO: 12.

[0022] In some embodiments, non-naturally occurring Nef proteins are linked to E3 ubiquitin ligases or fragments thereof via adapters.

[0023] In some embodiments, the connector comprises the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 25.

[0024] In some embodiments, non-naturally occurring Nef proteins are directly linked to E3 ubiquitin ligases or fragments thereof without a linker.

[0025] In some embodiments, the non-naturally occurring Nef protein is located at the N-terminus of an E3 ubiquitin ligase or a fragment thereof, or at the C-terminus of an E3 ubiquitin ligase or a fragment thereof.

[0026] In some embodiments, the fusion protein comprises an amino acid sequence of any one of SEQ ID NO: 6-10 or an amino acid sequence that is at least 90%, 95%, or 99% identical to an amino acid sequence of any one of SEQ ID NO: 6-10.

[0027] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 10.

[0028] In some embodiments, the fusion protein downregulates and / or degrades endogenous CD3ζ.

[0029] In one respect, this disclosure relates to nucleic acids containing a first nucleic acid sequence encoding the non-naturally occurring Nef protein or the fusion protein described herein.

[0030] In some embodiments, the nucleic acid further comprises a second nucleic acid sequence encoding an engineered receptor.

[0031] In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR).

[0032] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 or an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

[0033] In some embodiments, the nucleic acid further comprises a nucleic acid sequence encoding a protein marker.

[0034] In some embodiments, the protein marker is a mutant dihydrofolate reductase (DHFR) polypeptide having the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO: 20.

[0035] In some embodiments, the nucleic acid further comprises a nucleic acid sequence encoding a foreign cytokine or a fragment thereof.

[0036] In some embodiments, the exogenous cytokine or a fragment thereof is a membrane-bound interleukin 12β subunit (IL12p40) polypeptide having the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO: 21.

[0037] In some embodiments, each nucleic acid sequence is linked via a nucleic acid sequence encoding a cleavable adapter.

[0038] In some embodiments, the cuttable connector is P2A or T2A.

[0039] In one respect, this disclosure relates to a vector containing the nucleic acid described herein.

[0040] In one respect, this disclosure relates to engineered cells that contain the non-naturally occurring Nef protein described herein, the fusion protein described herein, the nucleic acid described herein, or the vector described herein.

[0041] In some embodiments, the engineered cell further comprises an engineered receptor.

[0042] In some embodiments, the engineered receptor is selected from the group consisting of engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), and T-cell antigen conjugates (TACs).

[0043] In some embodiments, the engineered receptor comprises:

[0044] (a) Extracellular ligand-binding domain;

[0045] (b) Transmembrane domains; and

[0046] (c) Intracellular signal transduction domain (ISD) comprising a chimeric signal transduction domain (CMSD) having an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence having SEQ ID NO: 15 or SEQ ID NO: 16.

[0047] In some embodiments, the engineered receptor further comprises a signal peptide located at the N-terminus of the extracellular ligand-binding domain.

[0048] In some embodiments, the extracellular ligand-binding domain of an engineered receptor binds to an antigen.

[0049] In some embodiments, the antigen is selected from the group consisting of: BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER -2, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, protease-3 (PR3), tyrosinase, survivin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, dentin 18.2, dentin 6, NKG2D, delta-like ligand 3 (DLL3), CD70, ADGRE2, FcRH5, NKp80, NKp30, NKG2A, CD229, and CS-1.

[0050] In some embodiments, the engineered receptor is a CAR.

[0051] In some embodiments, the intracellular signal transduction domain of the CAR further includes a co-stimulatory signal transduction domain, and the CAR further includes a hinge domain located between the C-terminus of the extracellular ligand binding domain and the N-terminus of the transmembrane domain.

[0052] In some embodiments, the engineered cell comprises a CAR containing an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

[0053] In some embodiments, the engineered cells further comprise a protein marker; optionally, the protein marker is a mutant DHFR polypeptide having the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO: 20.

[0054] In some embodiments, the engineered cells further comprise exogenous cytokines or fragments thereof.

[0055] In some embodiments, the exogenous cytokine or a fragment thereof is a membrane-bound IL12p40 polypeptide having the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO: 21.

[0056] In some embodiments, the engineered cells are selected from the group consisting of: T cells, αβT cells, γδT cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, and embryonic stem cells.

[0057] In some embodiments, the cell is a T cell.

[0058] In some embodiments, the engineered cells express a reduced level of CD3ζ compared to precursor T cells and / or express a reduced level of endogenous T cell receptor (TCR) compared to precursor T cells.

[0059] In some embodiments, engineered cells do not elicit a graft-versus-host disease (GvHD) response or elicit a reduced GvHD response in tissue-incompatible individuals compared to a GvHD response elicited by primary T cells isolated from the donor of precursor T cells derived from engineered cells.

[0060] In one respect, this disclosure relates to a method for producing engineered T cells, which includes introducing the nucleic acid or vector described herein into precursor cells.

[0061] In some embodiments, the method further includes introducing a second nucleic acid encoding a chimeric antigen receptor (CAR) into a precursor cell.

[0062] In one respect, this disclosure relates to a pharmaceutical composition comprising the engineered cells and pharmaceutically acceptable carriers described herein.

[0063] In one aspect, this disclosure relates to a method of treating a subject’s disease or disorder, the method comprising administering to the subject in need a therapeutically effective amount of the engineered cells or pharmaceutical composition described herein.

[0064] In one aspect, this disclosure relates to methods for downregulating and / or degrading target proteins in T cells derived from a subject, methods comprising introducing the nucleic acid or vector described herein into the T cells.

[0065] In some embodiments, the target protein is CD3ζ and / or TCR.

[0066] In one respect, this disclosure relates to chimeric antigen receptors (CARs), which include:

[0067] (1) An extracellular ligand-binding domain that binds to CD20, the extracellular ligand-binding domain comprising:

[0068] LCDR1 containing the amino acid sequence of SEQ ID NO: 26, LCDR2 containing the amino acid sequence of SEQ ID NO: 27, LCDR3 containing the amino acid sequence of SEQ ID NO: 28, HCDR1 containing the amino acid sequence of SEQ ID NO: 29, HCDR2 containing the amino acid sequence of SEQ ID NO: 30, and HCDR3 containing the amino acid sequence of SEQ ID NO: 31; wherein the CDR is determined according to the Kabat numbering scheme.

[0069] (2) Transmembrane domains, and

[0070] (3) Intracellular signal transduction domain (ISD) comprising a chimeric signal transduction domain (CMSD) having the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO: 16.

[0071] In some embodiments, the intracellular signal transduction domain further includes a co-stimulatory signal transduction domain.

[0072] In some embodiments, the extracellular ligand binding domain of the CAR comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 18.

[0073] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 14.

[0074] Other features and advantages of this disclosure will become apparent from the following detailed description, the accompanying drawings, and the claims. Attached Figure Description

[0075] Figure 1A and Figure 1B The expression of TCRαβ ( ) was shown in Jurkat cells containing truncated SIV Nef M116. Figure 1A ) and CD3ζ protein ( Figure 1B The results were obtained by flow cytometry analysis. M116-Jurkat, M708-Jurkat, M2237-Jurkat, and M2729-Jurkat cells overexpressed SIV Nef M116, SIV Nef M116 Trun1, SIV Nef M116 Trun2, and SIV Nef M116 Trun3, respectively. "Mimetic" refers to untransduced Jurkat cells used as a control.

[0076] Figure 2A and Figure 2B This demonstrates the expression of TCRαβ in CAR-positive (CAR+) Jurkat cells containing the truncated SIV Nef M116-TRIM21 Trun2 fusion protein. Figure 2A ) and CD3ζ protein ( Figure 2B The results were obtained by flow cytometry analysis. M2268-Jurkat, M2410-Jurkat, M2445-Jurkat, and M2444-Jurkat cells overexpressed the SIV NefM116-TRIM21 Trun2, SIV Nef M116 Trun1-TRIM21 Trun2, SIV Nef M116 Trun2-TRIM21 Trun2, and SIV Nef M116 Trun3-TRIM21 Trun2 fusion proteins, respectively. "Mimetic" refers to untransduced Jurkat cells used as a control.

[0077] Figure 3A and Figure 3B The results of flow cytometry analysis of CD20 CAR (CAR), TCRαβ, and MB12 (IL12p40) expression in M2664-T cells containing the fusion proteins SIV Nef M116 Trun3 and TRIM21 Trun2 are shown. “UnT” indicates untransduced T cells, which were used as controls.

[0078] Figure 4A and Figure 4B The results of flow cytometry and Western blot analysis of CD3ζ protein expression in M2664-T, CD3ζ KO-T, and UnT cells are shown.

[0079] Figure 5 The killing efficiency of M2664-T cells against the CD20-positive lymphoma cell line Raji.Luc was shown, with an E:T ratio of 20:1 or 10:1.

[0080] Figure 6 The selected sequences listed in this disclosure are shown. Detailed Implementation

[0081] This disclosure relates to non-naturally occurring Nef proteins (also known as mutant Nef proteins). Non-naturally occurring Nef proteins can be truncated Nef proteins, wherein the truncated Nef protein retains some or all of the functions of the wild-type Nef protein and has a smaller size than the wild-type Nef protein. Truncated Nef proteins are more advantageous due to their smaller size when co-expression with large proteins or several proteins is required; for example, "all-in-one" vectors can be more easily transferred to T cells. Truncated Nef proteins can be obtained from or derived from simian immunodeficiency virus (SIV) Nef isolates. Truncated Nef proteins can retain some or all of their functions in binding to CD3ζ and / or downregulating endogenous TCRs. Truncated Nef proteins can be obtained by deleting a fragment of the wild-type SIV Nef protein (e.g., a C-terminal fragment).

[0082] This disclosure also relates to a fusion protein comprising the non-naturally occurring Nef protein and E3 ubiquitin ligase or fragments thereof (i.e., a Nef fusion protein). This disclosure finds that expression of the fusion protein in cells (e.g., T cells) can induce the expression of a target protein (e.g., CD3ζ) in cells that are being specifically degraded. Without being bound by theory, the degradation of CD3ζ triggers downregulation of the TCR and interferes with TCR-mediated signal transduction, such as T cell activation or T cell proliferation. The E3 ubiquitin ligase or fragments thereof may be obtained from or derived from TRIM21.

[0083] In one aspect, this disclosure provides engineered cells that overexpress non-naturally occurring Nef proteins or Nef fusion proteins. These engineered cells can be further engineered to express engineered receptors, such as engineered TCRs (e.g., conventional engineered TCRs, chimeric TCRs (cTCRs)), TACs (T-cell antigen conjugates), TAC-like chimeric receptors, or CARs (e.g., antibody-based CARs, ligand / receptor-based CARs, or ACTRs (antibody-conjugated T-cell receptors)). The engineered receptor may comprise: (a) an extracellular ligand-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain (ISD) comprising a chimeric signaling domain (CMSD), wherein the CMSD comprises one or more ITAMs (“CMSD ITAM”). At least one of the CMSD ITAMs may not be CD3ζ-based. Therefore, the expression of the non-naturally occurring Nef proteins or Nef fusion proteins described herein has little effect on the expression of the engineered receptors. However, it should be understood that cells expressing non-naturally occurring Nef proteins or Nef fusion proteins do not need to contain any engineered receptors, or may contain engineered receptors that are not modified with ITAM, such as conventional CARs containing CD3ζISD.

[0084] In one aspect, this application provides for the prevention or mitigation of graft-versus-host disease (GvHD) associated with cell therapies (e.g., CAR-T or CAR-NT therapies). This disclosure is based in part on the finding that engineered immune cells overexpressing non-naturally occurring Nef proteins or Nef fusion proteins have reduced endogenous TCR levels and can be used in cell therapies. Downregulating endogenous TCR levels using non-naturally occurring Nef proteins or Nef fusion proteins can reduce GvHD and maintain the antitumor activity of adoptive cells. Non-naturally occurring Nef proteins or Nef fusion proteins can be overexpressed in adoptive cells such as chimeric antigen receptor T (CAR-T) cells.

[0085] In one aspect, this disclosure provides methods for downregulating endogenous TCR levels in immune cells by overexpressing non-naturally occurring Nef proteins or Nef fusion proteins. In another aspect, this disclosure provides methods for downregulating and / or degrading target proteins (e.g., CD3ζ and / or TCR) in T cells derived from a subject by overexpressing non-naturally occurring Nef proteins or Nef fusion proteins as described herein. In yet another aspect, this disclosure provides nucleic acids encoding non-naturally occurring Nef proteins and / or Nef fusion proteins.

[0086] As used herein, the term "overexpression" generally refers to any amount greater than or equal to the expression level exhibited by a reference standard. The terms "overexpression (overexpressing, overexpressed, and overexpression)" in this disclosure refer to a gene product or peptide expressed at a level higher than that expressed in a comparable host before gene alteration in the host cell or under defined conditions without gene alteration. If the host cell does not contain a given gene product, the gene product may be introduced into the host cell for expression; in this case, the term "overexpression" covers any detectable expression.

[0087] As used herein, the term "Nef protein" refers to a polypeptide derived from a wild-type Nef protein, an isotype of a Nef protein, or a functional variant thereof. A Nef protein can be a wild-type Nef protein (e.g., SIV Nef, HIV1 Nef, HIV2 Nef, and Nef homologs). A Nef protein can be a mutant Nef protein (e.g., a non-naturally occurring Nef protein) with one or more mutations (e.g., insertions, deletions, and / or substitutions). A non-naturally occurring Nef protein can be a mutant SIV Nef. A non-naturally occurring Nef protein can be a truncated Nef protein by deleting one or more segments of the wild-type Nef protein.

[0088] As used herein, the term "Nef fusion protein" refers to a polypeptide containing a Nef protein (e.g., a non-naturally occurring Nef protein) that has been fused to another protein. The Nef protein may fuse directly with the other protein or may be linked to the other protein via a linker. The other protein may be an E3 ubiquitin ligase or a fragment thereof (e.g., the TRIM21 protein).

[0089] As used herein, the term "TRIM21 protein" refers to a polypeptide derived from wild-type TRIM21 protein, an isotype of TRIM21 protein, or a functional variant thereof. TRIM21 is a member of the RING E3 ubiquitin ligase triple motif (TRIM) family, consisting of an N-terminal RING domain, a B-box domain, a coiled-coil dimerization domain, and a C-terminal PRYSPRY domain with E3 ubiquitin ligase activity. It recognizes the Fc domain and binds to immunoglobulin G, immunoglobulin A, and immunoglobulin M on antibody-labeled non-enveloped viral particles on infected cells. It then guides the viral particles to the proteasome via self-ubiquitination or cofactor ubiquitination, see, for example, Zeng, Jingwei et al., "Target-induced clustering activates Trim-Away of pathogens and proteins." Nature structural & molecular biology vol. 28,3 (2021): 278-289. The TRIM21 protein can be a wild-type TRIM21 protein (e.g., human TRIM21 protein). The TRIM21 protein can be a mutant TRIM21 protein with one or more mutations (e.g., insertion, deletion, and / or substitution). The TRIM21 protein can be a truncated TRIM21 containing a RING domain.

[0090] As used herein, a “vector” is any construct capable of delivering one or more nucleic acids of interest to a host cell when the vector is introduced into the host cell. An “expression vector” is capable of delivering one or more nucleic acids of interest and expressing them as encoded polypeptides in a host cell that has been introduced into the expression vector. Thus, in an expression vector, the nucleic acid of interest is operatively positioned for expression in the vector by being operatively linked to regulatory elements such as promoters, enhancers, and / or poly-A tails, which are located within the vector or in the genome of the host cell at or near the integration site of the nucleic acid of interest, such that the nucleic acid of interest will be translated in the host cell in which the expression vector has been introduced.

[0091] As used herein, the term "engineered receptor" refers to a foreign receptor (such as a CAR (e.g., antibody-based CAR, ligand / receptor-based CAR, or ACTR (antibody-conjugated T-cell receptor)), an engineered TCR (e.g., a conventional engineered TCR, a chimeric TCR (cTCR)), a T-cell antigen conjugate (TAC), or a TAC-like chimeric receptor) that retains its biological activity upon introduction into the T cells or engineered cells described herein. Biological activity includes, but is not limited to, the ability of the engineered receptor to specifically bind molecules (e.g., cancer antigens) and appropriately transduce downstream signals (such as inducing cell proliferation, cytokine production, and / or the execution of regulatory or cytolytic effector functions).

[0092] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically transplant one or more antigens onto immune effector cells, such as T cells. Some CARs are also referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." CARs may contain extracellular ligand-binding domains or extracellular antigen-binding domains, transmembrane domains, and intracellular signaling domains that are specific to one or more ligands or antigens, such as tumor antigens. "CAR-T cell" refers to a T cell that expresses a CAR. "CD20 CAR" refers to a CAR that has an extracellular binding domain specific to CD20.

[0093] As used herein, the term “cancer” refers to cells capable of autonomous growth. Examples of such cells include those exhibiting an abnormal state or condition characterized by rapid proliferative cell growth. The term is intended to encompass cancerous growth, such as tumors; carcinogenic processes, metastatic tissue, and malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. It also includes malignant tumors and neuroendocrine tumors (NETs) of various organ systems, such as respiratory, cardiovascular, renal, reproductive, hematologic, nervous, hepatic, gastrointestinal, and endocrine systems; and adenocarcinomas, including malignant tumors such as most colon cancers, renal cell carcinomas, prostate and / or testicular tumors, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNC), neuroendocrine prostate cancer (NEPC), pancreatic neuroendocrine tumors (PNET), gastrointestinal neuroendocrine carcinomas, and small bowel cancer. "Naturally occurring" cancer includes any cancer that is not experimentally induced by implanting cancer cells into a subject, and includes, for example, spontaneously occurring cancer, cancer caused by a patient's exposure to one or more carcinogens, cancer caused by the insertion of a transgenic oncogene or the knockout of a tumor suppressor gene, and cancer caused by infection (e.g., viral infection). The term "cancer" is generally accepted in the field as referring to a malignant tumor of epithelial or endocrine tissue. The term also includes carcinosarcoma, which comprises malignant tumors composed of both carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to cancer derived from glandular tissue or in which tumor cells form identifiable glandular structures. The term "sarcoma" is generally accepted in the field as referring to a malignant tumor derived from mesenchyme. The term "hematopoietic neoplastic disorder" includes diseases involving proliferative / tumor cells of hematopoietic origin. Hematopoietic neoplastic disorders can arise from myeloid, lymphoid, or erythroid lineages or their precursor cells.

[0094] When used in the context of target proteins in cells (e.g., endogenous CD3ζ or TCRs), the term “downregulation” refers to the downregulation, reduction, or decrease in the expression level of a target protein. Downregulation of a target protein can encompass the degradation of the target protein, such as through ubiquitination. It can also encompass the downregulation of the target receptor via, for example, internalization, stripping, capping, or other forms of receptor rearrangement changes on the cell surface. Endogenous TCR downregulation as described herein includes the downregulation of cell surface expression of endogenous TCRs (e.g., TCRα and / or TCRβ), and / or interference with TCR-mediated signal transduction, such as T cell activation or T cell proliferation. Endogenous TCR downregulation as described herein can be induced by the binding of Nef proteins to CD3ζ. Endogenous TCR downregulation as described herein can be induced by downregulating Nef fusion proteins of endogenous CD3ζ. The terms “down-modulate” and “down-regulate” are used interchangeably herein.

[0095] The terms “polypeptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. These terms also cover polymers of amino acids that have been naturally modified or modified by intervention; for example, by forming disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. This definition also includes, for example, polypeptides containing one or more amino acid analogs, including but not limited to non-natural amino acids, and other modifications known in the art. It should be understood that because the polypeptides disclosed herein may be based on antibodies or other members of the immunoglobulin superfamily, in some embodiments, a “polypeptide” may exist as a single chain or as two or more related chains.

[0096] The "percentage (%) amino acid sequence identity" and "homology" for peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, after alignment and, where necessary, the introduction of vacancies to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. Alignment to determine the percentage of amino acid sequence identity can be performed using publicly available calculator software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software, in various ways within the skill of the art. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full-length sequences being compared.

[0097] As used herein, the term “self” is intended to refer to any material originating from the same individual, which is subsequently reintroduced into that individual.

[0098] "Also-heterogeneous" refers to grafts that originate from different individuals of the same species.

[0099] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced using exogenous nucleic acids. This includes primary subject cells and their progeny.

[0100] It should be understood that the embodiments described herein include “consisting of embodiments” and / or “substantially consisting of embodiments”.

[0101] References to “about” values ​​or parameters in this document include (and describe) variations of that value or parameter itself. For example, a description involving “about X” includes a description of “X”.

[0102] The term “about XY” used in this article has the same meaning as “about X to about Y”.

[0103] When used in this document and the appended claims, the singular forms “a,” “or,” and “the” include plural references unless the context clearly indicates otherwise.

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. This document describes the methods and materials used in this disclosure; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification, including the definitions, shall prevail.

[0105] Nef protein and Nef fusion protein

[0106] Nef (negative regulatory factor) is a small 27-35 kDa myristylated protein encoded by primate lentiviruses, including human immunodeficiency virus (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV). Nef is primarily located in the cytoplasm, but also partially in the plasma membrane (PM), and is one of many proteins expressed by pathogens, known as virulence factors. Its function is to manipulate host cellular mechanisms to allow pathogen infection, survival, or replication.

[0107] T-cell receptor (TCR) signaling in response to antigen recognition plays a crucial role in the immune response and is regulated by many viral pathogens. The Nef protein of most SIVs and HIV-2 effectively prevents T-cell activation by removing the CD3 and TCR complex from the cell surface. CD3 is a key component of the TCR complex and is essential for intracellular signaling and cell surface expression of the TCR complex. Therefore, primate lentiviruses that downregulate CD3 prevent the formation of immune synapses between virus-infected CD4+ T cells and antigen-presenting cells (APCs) and inhibit T-cell activation.

[0108] A detailed review of Nef proteins and their functions can be found in Joas, Simone et al., "Nef-mediated CD3-TCR downmodulation dampens acute inflammation and promotes SIV immune evasion." Cell Reports 30.7 (2020): 2261-2274; and Laguette, Nadine et al., "Human immunodeficiency virus (HIV) type-1, HIV-2 and simian immunodeficiency virus Nef proteins." Molecular aspects of medicine 31.5 (2010): 418-433, which are included here in their full text by reference.

[0109] The Nef protein can be wild-type SIV Nef (UniProt accession number Q02840) having the amino acid sequence shown in SEQ ID NO: 1. The Nef protein can be a mutant with one or more mutations (e.g., insertions, deletions, and / or substitutions). The mutant Nef protein may contain alanine at position 178 and / or alanine at position 179 relative to the amino acid positions of wild-type SIV Nef. The mutant Nef protein may have an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2. The mutant Nef protein may have little downregulation of CD4 and CD28 expression.

[0110] In one aspect, this disclosure provides non-naturally occurring Nef proteins. Non-naturally occurring Nef proteins can be truncated Nef proteins. Non-naturally occurring Nef proteins can contain a deletion at the C-terminal segment of wild-type SIV Nef. Relative to the amino acid positions of wild-type SIV Nef, non-naturally occurring Nef proteins can contain a deletion of amino acids 207-223. Non-naturally occurring Nef proteins can contain a deletion of amino acids 207-223 as numbered in SEQ ID NO: 1 or their corresponding amino acids. Non-naturally occurring Nef proteins can consist of amino acids 1-206 as numbered in SEQ ID NO: 1 or their corresponding amino acids. Non-naturally occurring Nef proteins can contain a deletion at the C-terminal segment of mutant Nef. Relative to the amino acid positions of mutant Nef, non-naturally occurring Nef proteins can contain a deletion of amino acids 207-223. Non-naturally occurring Nef proteins can contain a deletion of amino acids 207-223 as numbered in SEQ ID NO: 2 or their corresponding amino acids. Non-naturally occurring Nef proteins may consist of amino acids 1-206 or their corresponding amino acids as numbered in SEQ ID NO: 2. Non-naturally occurring Nef proteins may have an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 4. In some cases, non-naturally occurring Nef proteins have an amino acid sequence identical to that of SEQ ID NO: 4. Non-naturally occurring Nef proteins may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations. Each mutation may be a deletion, insertion, or substitution. Non-naturally occurring Nef proteins may retain some or all of the functions of wild-type SIV Nef and have a smaller size than wild-type SIV Nef. Non-naturally occurring Nef proteins may retain some or all of the functions of mutant Nef proteins with the amino acid sequence shown in SEQ ID NO: 2, and have a smaller size than mutant Nef proteins. Non-naturally occurring Nef proteins may retain the function of binding to CD3ζ and / or downregulating endogenous TCRs. Non-naturally occurring Nef proteins may have little to no downregulation effect on CD4 and CD28 expression.

[0111] In one respect, this disclosure provides non-naturally occurring Nef proteins. Non-naturally occurring Nef proteins can be truncated Nef proteins. Relative to the amino acid positions of wild-type SIV Nef, non-naturally occurring Nef proteins can contain the deletion of amino acids 50-91. Non-naturally occurring Nef proteins can contain the deletion of amino acids 50-91 as numbered in SEQ ID NO: 1, or their corresponding amino acids. Non-naturally occurring Nef proteins can be composed of amino acids 1-49 and 92-223 as numbered in SEQ ID NO: 1, or their corresponding amino acids. Relative to the amino acid positions of mutant Nef, non-naturally occurring Nef proteins can contain the deletion of amino acids 50-91. Non-naturally occurring Nef proteins can contain the deletion of amino acids 50-91 as numbered in SEQ ID NO: 2, or their corresponding amino acids. Non-naturally occurring Nef proteins can be composed of amino acids 1-49 and 92-223 as numbered in SEQ ID NO: 2, or their corresponding amino acids. Non-naturally occurring Nef proteins may have an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 3. In some cases, non-naturally occurring Nef proteins have an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 3. Non-naturally occurring Nef proteins may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations. Each mutation may be a deletion, insertion, or substitution. Non-naturally occurring Nef proteins may retain some or all of the functions of wild-type SIV Nef and have a smaller size than wild-type SIV Nef. Non-naturally occurring Nef proteins may retain some or all of the functions of mutant Nef with the amino acid sequence shown in SEQ ID NO: 2 and have a smaller size than mutant Nef. Non-naturally occurring Nef proteins can retain their function of binding to CD3ζ and / or downregulating endogenous TCRs. Non-naturally occurring Nef proteins may have little downregulation effect on CD4 and CD28 expression.

[0112] In one aspect, this disclosure provides non-naturally occurring Nef proteins. Non-naturally occurring Nef proteins can be truncated Nef proteins. Relative to the amino acid positions of wild-type SIV Nef, non-naturally occurring Nef proteins can contain the deletion of amino acids 50-91 and 207-223. Non-naturally occurring Nef proteins can contain the deletion of amino acids 50-91 and 207-223, or their corresponding amino acids, as numbered in SEQ ID NO: 1. Non-naturally occurring Nef proteins can consist of amino acids 1-49 and 92-206, or their corresponding amino acids, as numbered in SEQ ID NO: 1. Relative to the amino acid positions of mutant Nef, non-naturally occurring Nef proteins can contain the deletion of amino acids 50-91 and 207-223. Non-naturally occurring Nef proteins can contain the deletion of amino acids 50-91 and 207-223, or their corresponding amino acids, as numbered in SEQ ID NO: 2. Non-naturally occurring Nef proteins may consist of amino acids 1-49 and 92-206, or their corresponding amino acids, as numbered in SEQ ID NO: 2. Non-naturally occurring Nef proteins may have an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 5. In some cases, non-naturally occurring Nef proteins have an amino acid sequence identical to that of SEQ ID NO: 5. Non-naturally occurring Nef proteins may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations. Each mutation may be a deletion, insertion, or substitution. Non-naturally occurring Nef proteins may retain some or all of the functions of wild-type SIV Nef and have a smaller size than wild-type SIV Nef. Non-naturally occurring Nef proteins may retain some or all of the functions of mutant Nef proteins with the amino acid sequence shown in SEQ ID NO: 2, and have a smaller size than mutant Nef proteins. Non-naturally occurring Nef proteins may retain the function of binding to CD3ζ and / or downregulating endogenous TCRs. Non-naturally occurring Nef proteins may have little to no downregulation effect on CD4 and CD28 expression.

[0113] The Nef protein described in this article (e.g., a non-naturally occurring Nef protein) can be expressed by an exogenous vector. The Nef protein can be generated in engineered cells (e.g., CAR-T cells) by introducing the exogenous vector via lentiviral infection. The Nef protein can be a truncated Nef protein. Exogenous vectors carrying truncated Nef proteins may be smaller and easier to transduce into cells compared to exogenous vectors carrying the full-length Nef protein. Truncated Nef proteins can increase lentiviral viral titers by more than 10%, more than 50%, more than 75%, more than 100%, more than 150%, more than 175%, more than 200%, more than 225%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, or more than 800% compared to the full-length Nef protein. The Nef protein described in this article can reduce endogenous TCR levels in engineered cells. The Nef protein can reduce GvHD.

[0114] In one respect, this disclosure provides a Nef fusion protein. The Nef fusion protein may comprise a Nef protein (e.g., the non-naturally occurring Nef protein shown herein) and an E3 ubiquitin ligase or a fragment thereof. The Nef protein and the E3 ubiquitin ligase or a fragment thereof may be directly linked to each other without a linker. The Nef protein and the E3 ubiquitin ligase or a fragment thereof may be linked to each other via a linker. The linker may comprise an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 25. The E3 ubiquitin ligase or a fragment thereof may be located at the N-terminus or C-terminus of the Nef protein.

[0115] The currently disclosed non-limiting exemplary E3 ubiquitin ligase of the Nef fusion protein is the TRIM21 protein. The TRIM21 protein may be a truncated TRIM21. The TRIM21 protein may be wild-type (wt) or mutant (mut). The TRIM21 protein may contain a RING domain derived from TRIM21. In some cases, the TRIM21 protein has an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12. In some cases, the TRIM21 protein has an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12. The TRIM21 protein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations. Each mutation can be a deletion, insertion, or substitution.

[0116] In one aspect, this disclosure provides a Nef fusion protein comprising a Nef protein (e.g., a mutant Nef protein) and a TRIM21 protein. The Nef protein may be a mutant having one or more mutations (e.g., insertions, deletions, and / or substitutions). The Nef protein may contain alanine at position 178 and / or alanine at position 179 relative to the amino acid positions of wild-type SIV Nef. The Nef protein may have the amino acid sequence shown in SEQ ID NO: 2. The TRIM21 protein may have the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12. The Nef fusion protein may have an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6. In some cases, the Nef fusion protein has an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 6. Nef fusion proteins can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations. Each mutation can be a deletion, insertion or substitution.

[0117] In one aspect, this disclosure provides a Nef fusion protein comprising a Nef protein (e.g., a non-naturally occurring Nef protein) and a TRIM21 protein. The non-naturally occurring Nef protein may contain the deletion of amino acids 207-223 relative to the amino acid positions of wild-type SIV Nef or its mutants. The non-naturally occurring Nef protein may contain the deletion of amino acids 207-223, or their corresponding amino acids, as numbered in SEQ ID NO: 1 and SEQ ID NO: 2. The non-naturally occurring Nef protein may consist of amino acids 1-206, or their corresponding amino acids, as numbered in SEQ ID NO: 1 and SEQ ID NO: 2. The non-naturally occurring Nef protein may have the amino acid sequence shown in SEQ ID NO: 4. The TRIM21 protein may have the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12. The Nef fusion protein may have an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8. In some cases, the Nef fusion protein has an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 8. The Nef fusion protein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations. Each mutation may be a deletion, insertion, or substitution.

[0118] In one aspect, this disclosure provides a Nef fusion protein comprising a Nef protein (e.g., a non-naturally occurring Nef protein) and a TRIM21 protein. The non-naturally occurring Nef protein may contain the deletion of amino acids 50-91 relative to the amino acid positions of wild-type SIV Nef or its mutants. The non-naturally occurring Nef protein may contain the deletion of amino acids 50-91, as numbered in SEQ ID NO: 1 and SEQ ID NO: 2, or the corresponding amino acids thereof. The non-naturally occurring Nef protein may consist of amino acids 1-49 and 92-223, as numbered in SEQ ID NO: 1 and SEQ ID NO: 2, or the corresponding amino acids thereof. The non-naturally occurring Nef protein may have the amino acid sequence shown in SEQ ID NO: 3. The TRIM21 protein may have the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12. The Nef fusion protein may have an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7. In some cases, the Nef fusion protein has an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 7. The Nef fusion protein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations. Each mutation may be a deletion, insertion, or substitution.

[0119] In one aspect, this disclosure provides a Nef fusion protein comprising a Nef protein (e.g., a non-naturally occurring Nef protein) and a TRIM21 protein. The non-naturally occurring Nef protein may contain the deletion of amino acids 50-91 and 207-223 relative to the amino acid positions of wild-type SIV Nef or its mutants. The non-naturally occurring Nef protein may contain the deletion of amino acids 50-91 and 207-223, or their corresponding amino acids, as numbered in SEQ ID NO: 1 and SEQ ID NO: 2. The non-naturally occurring Nef protein may consist of amino acids 1-49 and 92-206, or their corresponding amino acids, as numbered in SEQ ID NO: 1 and SEQ ID NO: 2. The non-naturally occurring Nef protein may have the amino acid sequence shown in SEQ ID NO: 5. The TRIM21 protein may have the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12. The Nef fusion protein may have at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO: 9 or SEQ ID NO: 10. The Nef fusion protein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations. Each mutation may be a deletion, insertion, or substitution.

[0120] In some cases, the Nef fusion protein has an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences in SEQ ID NO: 6-10. The Nef fusion protein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations. Each mutation may be a deletion, insertion, or substitution.

[0121] The Nef fusion protein may contain a linker between the Nef protein and the TRIM21 protein. The amino acid sequence may be at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 6-9.

[0122] Nef fusion proteins may contain Nef and TRIM21 proteins that bind directly to each other (without a linker). The amino acid sequence may be at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10.

[0123] The Nef fusion protein described in this article can be expressed by an exogenous vector. The Nef fusion protein can be generated in engineered cells (e.g., CAR-T cells) by introducing the exogenous vector via lentiviral infection. The Nef fusion protein can contain a truncated Nef protein. Compared to exogenous vectors carrying the full-length Nef protein, exogenous vectors carrying the truncated Nef protein may be smaller and easier to transduce into cells. Compared to the full-length Nef protein, the truncated Nef protein can increase the viral titer of lentivirus by more than 10%, more than 50%, more than 75%, more than 100%, more than 150%, more than 175%, more than 200%, more than 225%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, or more than 800%. The Nef fusion protein described in this article can induce the degradation of endogenous CD3ζ and / or reduce the level of endogenous TCR in engineered cells. The Nef fusion protein can reduce GVHD.

[0124] Also provided are nucleic acids (e.g., isolated nucleic acids) encoding any Nef protein described herein (e.g., non-naturally occurring Nef protein, truncated Nef protein) and / or any Nef fusion protein. Further provided are vectors (e.g., viral vectors, such as lentiviral vectors, bacterial expression vectors) containing nucleic acids encoding any Nef protein described herein and / or Nef fusion proteins. These vectors can be replaced with any vector described herein.

[0125] engineered cells

[0126] In one aspect, this disclosure provides engineered cells (e.g., engineered immune cells) with downregulated endogenous TCR levels. In another aspect, this disclosure provides engineered cells overexpressing Nef protein and / or Nef fusion protein.

[0127] In one aspect, this disclosure provides engineered cells that overexpress the Nef protein and / or Nef fusion protein described herein. Overexpression of the Nef protein and / or Nef fusion protein may be controlled by a non-natural regulatory element. This non-natural regulatory element may be an exogenous promoter. Engineered cells overexpressing the Nef protein and / or Nef fusion protein described herein may further express engineered receptors (e.g., CAR). The engineered cells described herein may express more than one engineered receptor (such as any combination of CAR, TCR, or TAC receptors). Engineered cells may further contain protein markers for screening purposes (e.g., DHFR L22F / F31S). Engineered cells may further contain a functional IL12 peptide (e.g., a membrane-bound IL12p40 peptide).

[0128] In one aspect, this disclosure provides engineered T cells (e.g., allogeneic T cells) comprising the Nef protein and / or Nef fusion protein described herein, wherein expression of the Nef protein and / or Nef fusion protein leads to downregulation of endogenous TCRs (e.g., TCRα and / or TCRβ) in the engineered T cells. Downregulation includes downregulating cell surface expression of endogenous TCRs. In some embodiments, cell surface expression of endogenous TCRs is downregulated by at least about 50%, 60%, 70%, 80%, 90%, 95%, or 97%. Endogenous CD3ζ expression may be downregulated by at least about 50%, 60%, 70%, 80%, 90%, or 95%. Endogenous CD3ζ expression may not be downregulated. Endogenous CD4 and / or CD28 expression may be downregulated. Endogenous CD4 and / or CD28 expression may not be downregulated. Nef protein and / or Nef fusion protein can downregulate the cell surface expression of endogenous TCRs, but not engineered receptors (such as engineered TCRs (e.g., conventional engineered TCRs, chimeric TCRs (cTCRs)), TACs, TAC-like chimeric receptors, or CARs (e.g., antibody-based CARs, ligand / receptor-based CARs, or ACTRs). In some embodiments, engineered receptors (such as engineered TCRs (e.g., conventional engineered TCRs, chimeric TCRs (cTCRs)), TACs, TAC-like chimeric receptors, or CARs (e.g., antibody-based CARs, ligand / receptor-based CARs, or ACTRs)) are downregulated by Nef protein and / or Nef fusion protein by up to about 50%, 40%, 30%, 20%, 10%, or 5%.

[0129] Examples of suitable non-natural regulatory elements include cytomegalovirus (CMV) promoters, early simian virus 40 (SV40) promoters, long terminal repeat (LTR) promoters of mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV), MoMuLV promoters, avian leukosis virus promoters, Epstein-Barr virus immediate early promoters, Raoult sarcoma virus promoters, EF-1α promoters, and human gene promoters (such as, but not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters). Inducible promoters are also contemplated as part of this disclosure. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operatively linked to it when such expression is desired, or turn off such expression when it is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0130] Engineered cells overexpressing Nef protein and / or Nef fusion protein may have multiple copies of the nucleic acid sequence encoding Nef protein and / or Nef fusion protein. Engineered cells may contain one, two, three, four, five or more copies of the coding sequence for Nef protein and / or Nef fusion protein.

[0131] The cells to be engineered can be obtained from, for example, humans or non-human animals. The cells to be engineered can be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pigs, or any other species. For example, cells can be obtained from humans, rats, or mice. Cells can be mouse lymphocytes and engineered (e.g., transduced) to express the Nef protein and / or Nef fusion protein described herein. Cells can be obtained from humans. Cells can be blood cells. Cells can be leukocytes (e.g., T cells), lymphocytes, or any other suitable blood cell type. Cells can be peripheral blood cells. Cells can be tumor-infiltrating lymphocytes (TILs). Cells can be T cells, B cells, NKT cells, or NK cells. Cells can be human peripheral blood mononuclear cells (PBMCs). Human PBMCs can be CD3+ cells. Human PBMCs can be CD8+ cells or CD4+ cells.

[0132] The cell may be a T cell. T cells may express one or more engineered receptors that recognize specific antigens on the surface of target cells. T cells can be obtained by various methods known in the art, such as the in vitro culture of T cells (e.g., tumor-infiltrating lymphocytes) isolated from a subject. Genetically engineered T cells can be obtained by transducing T cells (e.g., isolated from peripheral blood of a subject) with a vector (such as the vector provided herein). T cells may be CD4+ T cells, CD8+ T cells, or regulatory T cells. T cells may be T helper type 1 T cells and / or T helper type 2 T cells. T cells may be αβ-T cells. T cells expressing this receptor may be γδ-T cells. T cells may be central memory T cells. T cells may be effector memory T cells. T cells may be naive T cells.

[0133] The preparation of engineered immune cells may include one or more culture and / or preparation steps. The cells to be engineered can be isolated from a sample, such as a biological sample, for example, a sample obtained from or derived from a subject. The subject from whom the cells are isolated may have a disease or condition or require or be subject to cell therapy. The subject may be someone requiring a specific therapeutic intervention, such as adoptive cell therapy where the isolated, processed, and / or engineered cells are being prepared.

[0134] The cells can be stem cells, such as hematopoietic stem cells, embryonic stem cells, and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells can be primary cells, such as those isolated directly from the subject and / or isolated from the subject and frozen. Additional differentiation factors can be used to culture the stem cells to obtain the desired cell type (e.g., T cells).

[0135] Different cell types can be obtained via appropriate isolation methods. Isolation methods include separating different cell types based on the expression or presence of one or more specific molecules in the cells, such as surface markers, like surface proteins, intracellular markers, or nucleic acids. Any known isolation method based on such markers can be used. Isolation can be based on affinity or immunoaffinity. For example, in some aspects, isolation involves separating cells and cell populations based on the expression or expression level of one or more markers (typically cell surface markers), for example by incubation with antibodies or binding couplers that specifically bind to such markers, followed typically by a washing step and separation of cells bound to the antibody or binding coupler from those not bound to the antibody or binding coupler.

[0136] Such separation steps can be based on positive selection (where cells that bind to the reagent are retained for further use) and / or negative selection (where cells that do not bind to the antibody or binding coupler are retained). Both portions can be retained for further use. Negative selection can be particularly useful when no antibody is available for specific identification of cell types in a heterogeneous population, allowing for optimal separation based on markers expressed by cells other than the desired cell population.

[0137] Methods, nucleic acids, compositions, and kits (reagents) for expressing Nef proteins and / or Nef fusion proteins and / or engineered receptors, and / or for generating engineered immune cells are also provided. Genetic engineering typically involves introducing nucleic acids encoding Nef proteins and / or Nef fusion proteins into cells, such as through retroviral transduction, transfection, or transformation. Gene transfer can be accomplished by first stimulating cells, such as by combining them with stimuli that induce responses such as proliferation, survival, and / or activation (e.g., as measured by the expression of cytokines or activation markers), then transducing the activated cells, and expanding them in culture to a quantity sufficient for clinical application.

[0138] Recombinant nucleic acids can be transferred into cells using recombinant infectious viral particles, such as vectors derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). Recombinant nucleic acids can be transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors. Retroviral vectors can have long terminal repeats (LTRs), such as retroviral vectors derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), or spleen lesion-forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. Retroviruses can include those derived from any avian or mammalian cell source. Retroviruses are generally amphiphilic, meaning they are capable of infecting host cells of several species, including humans. The vector can be a lentiviral vector. In some embodiments, the vector contains any of the nucleic acids encoding the Nef protein and / or Nef fusion protein and / or engineered receptor described herein. Nucleic acids can be cloned into vectors using any molecular cloning method known in the art, including, for example, the use of restriction endonuclease sites and one or more selective markers. Recombinant nucleic acids can be transferred to T cells via electroporation. Recombinant nucleic acids can be transferred to T cells via transposition. Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection; tungsten particle-promoted microparticle bombardment; and strontium phosphate DNA coprecipitation. Many of these methods are described, for example, in WO 2019195486, which is incorporated herein by reference in its entirety. Prior to transduction, T cells can be pre-activated, for example, using anti-CD3 / CD28 particles, for approximately 12, 24, 36, 48, or 60 hours. Transduced T cells can be harvested on days 5, 6, 7, 8, 9, 10, 11, or 12 post-transduction.

[0139] Engineered immune cells can be used to treat a variety of disorders or diseases, such as cancer, as described in this article.

[0140] Engineered cells overexpressing Nef protein and / or Nef fusion protein may have reduced endogenous TCR levels compared to cells that do not express the Nef protein or Nef fusion protein described herein. Engineered cells overexpressing the Nef protein and / or Nef fusion protein described herein may not elicit a graft-versus-host disease (GvHD) response or may elicit a reduced GvHD response in tissue-incompatible individuals compared to cells that do not express the Nef protein or Nef fusion protein described herein.

[0141] The engineered cells described in this article can be eukaryotic cells, such as mammalian cells. Engineered cells can be human cells. Engineered cells can be horse, cow, mouse, sheep, dog, or cat cells. Engineered cells can be modified immune cells. Engineered cells can be T cells. Engineered cells can be NK cells. Engineered cells can be αβ T cells. Engineered cells can be γδ T cells. Engineered cells can be Vδ1 T cells.

[0142] For the individual receiving them, engineered cells can be autologous cells, syngeneic cells, allogeneic cells, or xenogeneic cells. Engineered cells can be modified by altering the major histocompatibility complex (MHC) profile, by inactivating β2-microglobulin to prevent the formation of functional class I MHC molecules, or by inactivating class II MHC molecules. Engineered cells can be autologous cells obtained from the recipient subject. Engineered cells can also be autologous T cells obtained from the recipient subject.

[0143] The engineered receptor can be a CAR. A CAR can comprise a polypeptide comprising, from its N-terminus to its C-terminus: a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB co-stimulatory signaling domain, and a primary intracellular signaling domain. In some embodiments, the CAR is a CD20 CAR. In some cases, the CAR comprises a polypeptide containing at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO: 13 or SEQ ID NO: 14.

[0144] Nef proteins may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 3-5.

[0145] The Nef fusion protein may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 6-10.

[0146] Engineered cells can overexpress Nef protein and / or Nef fusion protein and express CAR (CAR armored with Nef protein and / or Nef fusion protein). Engineered cells can be modified CAR-T cells (CAR-T cells armored with Nef protein and / or Nef fusion protein).

[0147] Engineered cells can contain peptides containing CAR and Nef proteins. The peptide can contain, from the N-terminus to the C-terminus, the following components: Nef protein, 2A adapter, CD8α signal peptide, extracellular antigen-binding domain, CD8α hinge domain, CD8α transmembrane domain, 4-1BB co-stimulatory signal transduction domain, and primary intracellular signal transduction domain.

[0148] Engineered cells can contain peptides containing CAR and Nef fusion proteins. The peptides can include, from the N-terminus to the C-terminus, the following components: Nef fusion protein, 2A adapter, CD8α signal peptide, extracellular antigen-binding domain, CD8α hinge domain, CD8α transmembrane domain, 4-1BB co-stimulatory signal transduction domain, and primary intracellular signal transduction domain.

[0149] Engineered cells overexpressing the Nef protein and / or Nef fusion protein described herein may have reduced endogenous TCR levels compared to cells that do not express the Nef protein or Nef fusion protein described herein. TCRαβ expression in engineered cells can be determined by flow cytometry (FACS) (e.g., as described in Example 1). Engineered cells may have TCRαβ positivity rates greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, or greater than 90%. Engineered cells may have TCRαβ positivity rates less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, or less than 90%. In some cases, engineered cells have a TCRαβ positivity rate of 10%-50%, 10%-40%, or 10%-40%.

[0150] Engineered cells overexpressing the Nef protein and / or Nef fusion protein described herein may have reduced endogenous TCR levels compared to cells that do not express the Nef protein or Nef fusion protein described herein. CD3ζ expression in engineered cells can be determined by flow cytometry (FACS) (e.g., as described in Example 1). Engineered cells may have CD3ζ positivity rates greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, or greater than 90%. Engineered cells may have CD3ζ positivity rates less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, or less than 90%. In some cases, engineered cells have a CD3ζ positivity rate of 98%-100%.

[0151] Engineered cells overexpressing the Nef protein and / or Nef fusion protein described herein may have reduced endogenous TCR levels compared to cells that do not express the Nef protein or Nef fusion protein described herein. TCRαβ expression in engineered cells can be determined by flow cytometry (FACS) (e.g., as described in Example 2). In CAR-positive cells, engineered cells may have a TCRαβ positivity rate greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, or greater than 90%. In CAR-positive cells, engineered cells can have TCRαβ positivity rates of less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, or less than 90%. In some cases, engineered cells in CAR-positive cells have TCRαβ positivity rates of 1%-10%, 1%-5%, or 3%-5%.

[0152] Engineered cells overexpressing the Nef protein and / or Nef fusion protein described herein may have reduced endogenous TCR levels compared to cells that do not express the Nef protein or Nef fusion protein described herein. CD3ζ expression in engineered cells can be determined by flow cytometry (FACS) (e.g., as described in Example 2). In CAR-positive cells, engineered cells may have CD3ζ positivity rates greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, or greater than 90%. In CAR-positive cells, engineered cells may have CD3ζ positivity rates less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, or less than 90%. In some cases, engineered cells in CAR-positive cells have a CD3ζ positivity rate of 1%-30%, 1%-20%, 10%-20%, or 12%-17%.

[0153] The expression of CAR and Nef protein and / or Nef fusion protein in engineered cells can be determined by flow cytometry (FACS) (e.g., as described in Example 2). Engineered cells can have CAR positivity rates greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. Engineered cells can also have CAR positivity rates less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. In some cases, engineered cells have CAR positivity rates of 20%–50%, 20%–40%, or 25%–40%.

[0154] Flow cytometry (FACS) can be used to determine the positivity rates of Nef protein and Nef fusion protein in engineered cells. Engineered cells can have Nef protein or Nef fusion protein positivity rates greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. Engineered cells can also have Nef protein or Nef fusion protein positivity rates less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. In some cases, engineered cells have a positivity rate of 10%-50%, 10%-40%, or 15%-40% for Nef protein or Nef fusion protein.

[0155] Engineered cells can be enriched by cell sorting (e.g., as described in Example 3). After enrichment, the percentage of TCRαβ-negative and CAR-positive cells can be greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, or greater than 90%. After enrichment, the percentage of TCRαβ-negative and CAR-positive cells can be less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, or less than 90%. After enrichment, the percentage of TCRαβ-negative and CAR-positive cells can be 50%-100%, 70%-100%, or 80%-100%.

[0156] Engineered cells can be enriched by cell sorting (e.g., as described in Example 3). After enrichment, the percentage of MB12-positive and CAR-positive cells can be greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, or greater than 90%. After enrichment, the percentage of MB12-positive and CAR-positive cells can be less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, or less than 90%. After enrichment, the percentage of MB12-positive and CAR-positive cells can be 50%-100%, 70%-100%, or 75%-100%.

[0157] Engineered cell populations, compositions containing such cells and / or enriched with such cells may comprise at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a higher percentage of the total cells or a certain type of cells (such as T cells, CD8+ or CD4+ cells) in the composition.

[0158] After enrichment, CD3ζ expression in engineered cells can be determined by flow cytometry (FACS) (e.g., as described in Example 3). After enrichment, engineered cells can have CD3ζ positivity rates greater than 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. After enrichment, engineered cells can have CD3ζ positivity rates less than 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. After enrichment, engineered cells can have a CD3ζ positivity rate of 0.1%-10%, 0.1%-5%, 0.1%-2%, or 0.2%-2%.

[0159] After enrichment, CD3ζ expression in engineered cells can be determined by Western blotting (e.g., as described in Example 3). After enrichment, engineered cells can have CD3ζ positivity rates greater than 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. After enrichment, engineered cells can have CD3ζ positivity rates less than 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. After enrichment, engineered cells can have a CD3ζ positivity rate of 0.1%-10%, 0.1%-5%, 0.1%-2%, or 0.2%-2%.

[0160] On days 0, 1, 2, 3, 4, or 5 post-transduction, the viability of the transduced T cells may be at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%. On days 0, 1, 2, 3, 4, or 5 post-transduction (e.g., day 5), the viability of the transduced T cells may be at least or about 80%, at least or about 90%, at least or about 100%, at least or about 110%, or at least or about 120% compared to the viability of untransduced T cells.

[0161] On days 0, 1, 2, 3, 4, or 5 post-transduction, the T cell expansion fold can be at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times. On days 0, 1, 2, 3, 4, or 5 post-transduction (e.g., day 5), the T cell expansion fold of the transduced T cells can be at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, or at least or about 90% higher than that of the untransduced T cells.

[0162] Engineered cells (e.g., CAR-T cells) can kill tumor cells. The cytotoxicity of engineered cells against tumor cells (e.g., Raji cells) can be determined by in vitro cytotoxicity assays (e.g., as described in Example 3). Engineered cells (e.g., CAR-T cells) can be co-cultured with target cells for at least or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 16 hours, 18 hours, 1 day, 2 days, 3 days, or longer, allowing the engineered cells (e.g., CAR-T cells) to be activated. The effector cell:target cell (E:T) ratio can be 0.3:1, 0.5:1, 1:1, 2:1, 2.5:1, 5:1, 10:1, or 20:1. Compared to untransduced T cells, engineered cells can exhibit cytotoxicity exceeding 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. Cytotoxicity may be specific against target cells expressing the target antigen (e.g., CD20). Engineered cells may induce little to no cytotoxicity or induce background cytotoxicity against target cells not expressing the target antigen (e.g., cells not expressing CD20).

[0163] Engineered cells can also express protein markers. These protein markers can be dihydrofolate reductase (DHFR) proteins (e.g., mutant DHFR proteins). DHFR proteins can have an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20. DHFR proteins can serve as genetic markers for specifying trimethoprim resistance (TmpR).

[0164] Engineered cells can also express exogenous cytokines or fragments thereof. Exogenous cytokines or fragments thereof may be membrane-bound interleukin-12 β subunit (IL12p40) protein. The IL12p40 protein may have an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21.

[0165] Cells used for engineering can be isolated from a sample, such as a biological sample, for example, a sample obtained from or derived from a subject. The subject from whom cells are isolated can be a subject suffering from a disease or condition, requiring cell therapy, or to whom cell therapy will be administered. The subject can be someone requiring a specific therapeutic intervention, such as adoptive cell therapy where cells are being isolated, processed, and / or engineered.

[0166] Cells can be primary cells, such as primary human cells. Samples can include tissues, fluids, and other samples taken directly from the subject, as well as samples produced by one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples can be samples obtained directly from biological sources or processed samples. Biological samples include, but are not limited to, bodily fluids (such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat), tissue and organ samples, including processed samples derived therefrom.

[0167] Engineered cell precursor T cells may express reduced levels of CD3ζ and / or reduced levels of endogenous T cell receptors (TCRs) compared to precursor T cells. Engineered cells may not elicit a graft-versus-host disease (GvHD) response or elicit a reduced GvHD response in tissue-incompatible individuals compared to a GvHD response induced by primary T cells isolated from the donor of the precursor T cells derived from engineered cells.

[0168] engineered receptors

[0169] One aspect of this disclosure provides engineered cells (e.g., immune cells) that overexpress the Nef protein and / or Nef fusion protein described herein and express engineered receptors. The engineered receptor may comprise an extracellular ligand-binding domain or an extracellular antigen-binding domain that specifically binds to an antigen (e.g., a tumor antigen), a transmembrane domain, and an intracellular signal transduction domain. The engineered receptor may comprise a signal peptide located at the N-terminus of the extracellular ligand-binding domain. The engineered receptor may be encoded by a heteropolynucleotide operatively linked to a promoter, such as a constitutive or inducible promoter. Exemplary engineered receptors include, but are not limited to, chimeric antigen receptors (CARs), engineered T-cell receptors (TCRs), and T-cell antigen-coupled receptors (TACs).

[0170] Engineered receptors can be monovalent and monospecific. Engineered receptors can be multivalent and monospecific. Engineered receptors can be multivalent and multispecific.

[0171] Engineered receptors may comprise: (a) an extracellular ligand-binding domain, (b) a transmembrane domain (e.g., derived from CD8α), and (c) an intracellular signaling domain (ISD) containing a chimeric signaling domain (CMSD). The CMSD may contain one or more signaling motifs referred to as immune receptor tyrosine-based activation motifs or ITAMs. Multiple ITAMs may be linked by one or more adapters. Multiple ITAMs may be directly linked to each other. ITAMs may be derived from proteins selected from the group consisting of: CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγR IIa, DAP10, and DAP12. In some embodiments, the engineered receptor has an intracellular signaling domain derived from CD3ζ. In some embodiments, the engineered receptor does not have any sequence derived from CD3ζ (e.g., human CD3ζ). In some embodiments, at least one ITAM in the engineered receptor is not derived from CD3ζ. In some embodiments, at least one ITAM in the engineered receptor is not ITAM1 or ITAM2 of CD3ζ. CMSD may comprise ITAM010 or ITAM045. ITAM010 has the amino acid sequence shown in SEQ ID NO: 15. ITAM045 has the amino acid sequence shown in SEQ ID NO: 16. ITAM010 and ITAM045 are disclosed in PCT application publication number WO 2021037221 (the entire contents of which are incorporated herein by reference).

[0172] The antigens for engineered receptors can be tumor antigens selected from the following groups: BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, hTERT, IL-13Rα2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE- A1, mesothelin, MAGEA3, p53, MART1, GP100, protease-3 (PR3), tyrosinase, survivin, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, IGF1R, EGFR, EGFR-VIII, dentin 18.2, dentin 6, NKG2D, delta-like ligand 3 (DLL3), CD70, CS-1, c-Met, glycolipid F77, PD-L1, PD-L2, ADGRE2, FcRH5, NKp80, NKp30, NKG2A, CD229, and other clinically significant tumor antigens, and combinations thereof. Antigens can be CD20. Tumor antigens can be derived from intracellular proteins of tumor cells. Tumor antigens can be expressed on the surface of tumor cells. Many TCRs specific to tumor antigens, including tumor-associated antigens, have been described, such as the NY-ESO-1 cancer-testis antigen, the p53 tumor suppressor antigen, and TCRs targeting tumor antigens in melanoma (e.g., MARTI, gp 100), leukemia (e.g., WT1, minor histocompatibility antigen), and breast cancer (e.g., HER2, NY-BR1).

[0173] The engineered receptors described herein can be chimeric antigen receptors (CARs). Many chimeric antigen receptors are known in the art and can be adapted to the engineered cells described herein. CARs can also be constructed to be specific for any cell surface marker by using, for example, antigen-binding fragments of antibody molecules or variable domains of antibodies.

[0174] The CAR disclosed herein may include an extracellular domain, a transmembrane region, and an intracellular signal transduction domain, wherein the extracellular domain includes at least one antigen-binding domain that specifically binds at least one tumor antigen.

[0175] Intracellular signaling domains can generate signals that promote immune effector functions in CAR-containing cells (e.g., CAR-T cells). Immune effector functions or immune effector responses refer to functions or responses of immune effector cells that enhance or promote immune attack on target cells. For example, immune effector functions or responses can refer to the properties of T or NK cells that promote the killing of target cells or inhibit the growth or proliferation of target cells. Examples of immune effector functions, such as in CAR-T cells, include cytolytic activities (such as antibody-dependent cytotoxicity or ADCC) and helper activities (such as cytokine secretion). Intracellular signaling domains can generate signals that promote the proliferation and / or survival of CAR-containing cells. Signaling domains of naturally occurring molecules can comprise the entire intracellular (or cytoplasmic) portion of the molecule or the entire natural intracellular signaling domain, or fragments or derivatives thereof.

[0176] The intracellular signaling domain of a CAR may contain one or more (such as any one of 1, 2, 3 or more) co-stimulatory signaling domains. A “co-stimulatory signaling domain” can be the intracellular portion of a co-stimulatory molecule. The term “co-stimulatory molecule” refers to a homologous binding partner on an immune cell (such as a T cell) that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of the immune cell, such as, but not limited to, proliferation and survival. Co-stimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that contribute to an efficient immune response. Co-stimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), and activating NK cell receptors. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Other examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, L... FA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83.

[0177] A CAR may contain a single costimulatory signaling domain. A CAR may contain two or more costimulatory signaling domains. Intracellular signaling domains may contain a functional primary intracellular signaling domain (e.g., the chimeric signaling domain (CMSD) shown herein) and one or more costimulatory signaling domains. A CAR may not contain a functional primary intracellular signaling domain (such as CD3ζ). A CAR may contain an intracellular signaling domain consisting of or substantially consisting of one or more costimulatory signaling domains. A CAR may contain an intracellular signaling domain consisting of a nonfunctional or attenuated primary intracellular signaling domain (such as mutant CD3ζ) and one or more costimulatory signaling domains, or substantially consisting of a nonfunctional or attenuated primary intracellular signaling domain (such as mutant CD3ζ) and one or more costimulatory signaling domains. After the antigen-binding domain binds to the tumor antigen, the co-stimulatory signaling domain of the CAR can transduce signals to enhance the proliferation, survival, and differentiation of CAR-equipped modified immune cells (such as T cells) and inhibit activation-induced cell death. One or more co-stimulatory signaling domains can derive from one or more molecules selected from the group consisting of: CD27, CD28, 4-1BB (i.e., CD137), OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

[0178] The antigen-binding domain of a CAR may contain one or more (such as 1, 2, 3, 4, 5, 6 or more) antibodies or antibody fragments, which may be selected from scFv, Fv, Fab, (Fab′)2, mini antibodies, biantibodies, single-domain antibodies (sdAb), or V. H The H domain. The antigen-binding domain of a CAR may contain an extracellular portion of a ligand or receptor that specifically binds to a tumor antigen. CARs can be monospecific, bispecific, or multispecific. The antigen-binding domain of a CAR can specifically bind to a single tumor antigen. The antigen-binding domain of a CAR can bind to two or more tumor antigens. Engineered receptors (e.g., CARs) can redirect the specificity of engineered cells by expressing chimeric antigen receptors (CARs) or TCRs on these cells.

[0179] The transmembrane region of a CAR may contain transmembrane regions selected from the following: α, β, or ζ chains of the T cell receptor; CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154; KIRDS2, OX40, CD2, CD27; LFA-1 (CD11a, CD18); ICOS (CD278); 4-1BB (CD137); GITR; CD40; BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 (KLRF1); CD160; CD19; IL-2Rβ; IL-2Rγ; IL-7R. a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITG AX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT Transmembrane regions of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. The transmembrane domains of a CAR can be CD4, CD3, CD8α, or CD28 transmembrane domains. The transmembrane region of a CAR may contain a CD8α transmembrane domain.

[0180] Extracellular domains can connect to transmembrane domains via hinge domains. The hinge domain can be a CD8α hinge domain.

[0181] CARs can also contain signal peptides (SPs), such as the CD8α signal peptide.

[0182] Many CARs targeting different tumor antigens have been widely disclosed in the art, such as CD19 CARs or BCMACARs. The extracellular antigen-binding domain of a CD19 CAR may be or include a CD19-binding fragment (e.g., FMC63, SJ25C1, or those disclosed in various patents such as WO 2022 / 012683). BCMA CARs have also been well described, with relevant patents including, but not limited to, WO 2016 / 014789, WO 2016 / 014565, WO 2013 / 154760, and WO 2018 / 028647. The extracellular antigen-binding domain of a BCMACAR may be or include a BCMA-binding fragment. The BCMA-binding fragment may bind to one or more epitopes on BCMA. A BCMA CAR may be a bivalent CAR comprising two anti-BCMA sdAbs targeting the same or different BCMA epitopes.

[0183] The CAR can be a CD20 CAR. A CD20 CAR can be monospecific. A CD20 CAR can be bispecific or bivalent. The extracellular antigen-binding domain of a CD20 CAR can be or include one or more CD20-binding moieties. The CD20-binding moieties can bind to one or more epitopes on CD20. A CD20 CAR can be a bivalent CAR containing two anti-CD20 sdAbs (e.g., VHH domains) targeting the same or different CD20 epitopes. Various antigen-binding domain sequences can be used as the antigen-binding domain of a CD20 CAR.

[0184] The CD20 CAR may comprise, from the N-terminus to the C-terminus, the following: a CD8α signal peptide, an extracellular antigen-binding domain containing anti-CD20 scFv, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB co-stimulatory signal transduction domain, and a primary intracellular signal transduction domain. The anti-CD20 scFv may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to that of SEQ ID NO: 17 or SEQ ID NO: 18.

[0185] The CD20 CAR may comprise an anti-CD20 scFv comprising: HCDR1 (heavy chain CDR1), which comprises the amino acid sequence of SEQ ID NO: 29 or a variant thereof comprising up to about 3 amino acid substitutions; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 30 or a variant thereof comprising up to about 3 amino acid substitutions; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 31 or a variant thereof comprising up to about 3 amino acid substitutions; and LCDR1 (light chain CDR1), which comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof comprising up to about 3 amino acid substitutions; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof comprising up to about 3 amino acid substitutions; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 28 or a variant thereof comprising up to about 3 amino acid substitutions. The CDR sequences may be determined according to a well-known numbering system. In some embodiments, the CDR is based on the Kabat number.

[0186] The primary intracellular signal transduction domain (e.g., ITAM010, ITAM045) may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to that of SEQ ID NO: 15 or SEQ ID NO: 16.

[0187] The CD20 CAR may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to those in SEQ ID NO: 13 or SEQ ID NO: 14. The CD20 CAR can specifically bind to CD20-positive tumor cells (e.g., Raji cells).

[0188] In one aspect, this disclosure provides a CAR comprising (1) an extracellular ligand-binding domain that binds to CD20, the extracellular ligand-binding domain comprising: LCDR1 comprising the amino acid sequence of SEQ ID NO: 26, LCDR2 comprising the amino acid sequence of SEQ ID NO: 27, LCDR3 comprising the amino acid sequence of SEQ ID NO: 28, HCDR1 comprising the amino acid sequence of SEQ ID NO: 29, HCDR2 comprising the amino acid sequence of SEQ ID NO: 30 and HCDR3 comprising the amino acid sequence of SEQ ID NO: 31; (2) a transmembrane domain; and (3) an intracellular signal transduction domain (ISD) comprising a chimeric signal transduction domain (CMSD) having the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO: 16.

[0189] The engineered receptor can be a modified T-cell receptor or an engineered T-cell receptor. The engineered TCR can be specific to any tumor antigen shown herein. Any TCR known in the art can be used. The TCR can have enhanced affinity for the tumor antigen. Exemplary TCRs and methods for introducing TCRs into immune cells have been described, for example, in U.S. Patent No. 5,830,755 and Kessels et al., Immunotherapy through TCR gene transfer. Nat. Immunol. 2, 957-961 (2001), which are incorporated herein by reference in their entirety.

[0190] The TCR receptor complex is an octamer formed by the variable TCR receptor α and β chains (or γ and δ chains in the case of γδ T cells) with three dimeric signaling modules CD3δ / ε, CD3γ / ε, and CD247 (the CD3ζ chain of the T cell surface glycoprotein) ζ / ζ or ζ / η. Ionizable residues in the transmembrane region of each subunit form an interacting polar network that holds the complex together. The TCR complex has the function of activating the signal transduction cascade in T cells.

[0191] Engineered receptors can be engineered TCRs comprising one or more T-cell receptor (TCR) fusion proteins (TFPs). Exemplary TFPs have been described, for example, in US 20170166622A1, which is incorporated herein by reference in its entirety. A TFP may comprise an extracellular domain of a TCR subunit comprising an extracellular domain or a portion thereof of a protein selected from the group consisting of: a TCRα chain, a TCRβ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, a functional fragment thereof, and an amino acid sequence having at least one but no more than 20 modified amino acids. A TFP may comprise a transmembrane region comprising a transmembrane region of a protein selected from the group consisting of: a TCRα chain, a TCRβ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, a functional fragment thereof, and an amino acid sequence having at least one but no more than 20 modified amino acids. TFP may contain a transmembrane region comprising a transmembrane region of a protein selected from the group consisting of: TCRα chain, TCRβ chain, TCRζ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, their functional fragments, and their amino acid sequences having at least one but no more than 20 modifications.

[0192] TFP may comprise a TCR subunit and an antigen-binding domain, the TCR subunit comprising at least a portion of the extracellular domain of the TCR and an intracellular domain of the TCR containing a stimulatory domain from the intracellular signaling domain of CD3ε; wherein the TCR subunit is operatively linked to the antigen-binding domain, and wherein the TFP is incorporated into the TCR when expressed in T cells.

[0193] Engineered receptors can be T-cell antigen-coupled (TAC) receptors. For example, an exemplary TAC receptor has been described in US20160368964A1, which is incorporated herein by reference. A TAC may comprise an antigen-binding domain, a TCR-binding domain that specifically binds to a protein associated with the TCR complex, and a T-cell receptor signaling domain. The antigen-binding domain may be an antibody fragment that specifically binds to a tumor antigen, such as scFv or VHH. The antigen-binding domain may be a designed ankyrin repeat (DARPin) polypeptide. The tumor antigen may be any antigen shown herein. The protein associated with the TCR complex may be CD3, such as CD3E. The TCR-binding domain may be a single-chain antibody, such as scFv or VHH. HH. The TCR-binding domain may be derived from UCHT1. The TAC receptor may include a cytoplasmic domain and a transmembrane domain. The T cell receptor signaling domain may include a cytoplasmic domain derived from a TCR co-receptor. Exemplary TCR co-receptors include, but are not limited to, CD4, CD8, CD28, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. The TAC receptor may include a transmembrane domain and a cytoplasmic domain derived from CD4. The TAC receptor may include a transmembrane domain and a cytoplasmic domain derived from CD8 (such as CD8α).

[0194] T cell co-receptors are expressed as membrane proteins on T cells. They can stabilize the TCR:peptide:MEC complex and promote signal transduction. Two subtypes of T cell co-receptors, CD4 and CD8, exhibit strong specificity for specific MEC classes. The CD4 co-receptor stabilizes only the TCR:MEC II complex, while the CD8 co-receptor stabilizes only the TCR:MEC I complex. Differential expression of CD4 and CD8 across different T cell types leads to distinct functional subsets of T cells. CD8+ T cells are cytotoxic T cells.

[0195] Engineered receptors (such as CARs, engineered TCRs, or TACs) can target one or more tumor antigens. Tumor antigens are proteins produced by tumor cells that can trigger an immune response, particularly a T-cell-mediated immune response. The choice of target antigen will depend on the specific type of cancer to be treated. Exemplary tumor antigens include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, muta-hsp70-2, M-CSF, prostate enzymes, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostaglandins, PSMA, HER2 / neu, survival proteins and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, liver glycoside B2 (ephrinB2), CD22, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0196] Tumor antigens can contain one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express a number of proteins that can be used as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and gp100 in melanoma, and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the transformation-related molecular group, such as the oncogene HER2 / Neu / ErbB-2. Another group of target antigens is oncoemulsification antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotype immunoglobulins constitute the true tumor-specific immunoglobulin antigens unique to the individual tumor. B-cell differentiation antigens (such as CD19, CD20, and CD37) are other candidates for target antigens in B-cell lymphomas.

[0197] Tumor antigens can be tumor-specific antigens (TSA) or tumor-associated antigens (TAAs). TSAs are specific to tumor cells and are not present on other cells in the body. TAA-associated antigens are not specific to tumor cells; instead, they are also expressed on normal cells under conditions that do not induce immune tolerance to the antigen. Antigen expression on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs can be antigens expressed on normal cells during embryonic development (when the immune system is immature and unable to respond), or they can be antigens that are normally present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0198] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens, such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER2 / neu; unique tumor antigens resulting from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA 242, CA-50, CAM43, CD68, P1, CO-029, FGF-5, G250, Ga733, EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90, Mac-2 binding protein, cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0199] Nucleic acid

[0200] This disclosure provides (i) nucleic acids encoding Nef proteins and / or Nef fusion proteins, and (ii) nucleic acids encoding engineered receptors (e.g., CARs, TACs, or engineered TCRs). The nucleic acids disclosed herein may contain nucleic acid sequences encoding any one of the Nef proteins, Nef fusion proteins, and / or engineered receptors disclosed herein. The nucleic acids may encode both (1) the Nef protein or the Nef fusion protein and (2) the CAR. The nucleic acids may contain one, two, three, four, five, or more copies of the coding sequences for the Nef protein and / or the Nef fusion protein. Expression of the Nef protein and / or the Nef fusion protein may be controlled by non-natural regulatory elements.

[0201] The nucleic acid disclosed herein may comprise a first nucleic acid sequence and a second nucleic acid sequence. The first nucleic acid may be upstream of or downstream of the second nucleic acid. The first and second nucleic acid sequences may be separated by a linker. The linker used in this disclosure allows multiple proteins encoded by the same nucleic acid sequence (e.g., a polycistronic or bicistronic sequence), which are translated into polyproteins that dissociate into individual protein components. The nucleic acid may comprise a first nucleic acid sequence, a linker, and a second nucleic acid sequence from its 5' to 3' end. Alternatively, the nucleic acid may comprise a second nucleic acid sequence, a linker, and a first nucleic acid sequence from its 5' to 3' end. The first nucleic acid sequence may encode the Nef protein and / or Nef fusion protein described herein, and the second nucleic acid sequence may encode the CAR described herein.

[0202] The adapter may contain a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, “internal ribosome entry site” or “IRES” refers to an element that facilitates direct entry of an internal ribosome into a protein-coding region of a start codon (such as ATG), thereby leading to cap-independent translation of the gene. Various internal ribosome entry sites are known to those skilled in the art, including but not limited to IRES derived from viral or cellular mRNA sources such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES derived from, for example, heart virus, rhinovirus, foot-and-mouth disease virus, HCV, Friend mouse leukemia virus (FrMLV), and Moloney mouse leukemia virus (MoMLV). Those skilled in the art will be able to select an appropriate IRES.

[0203] Linkers may contain nucleic acid sequences encoding self-cleaving peptides. As used herein, a “self-cleaving peptide” or “2A linker” refers to an oligopeptide that allows multiple proteins to be encoded as polyproteins that dissociate into component proteins upon translation. The use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A linkers are known to those skilled in the art, including but not limited to those found in members of the Picornaviridae virus family, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV0), Thosea asignavirus (TaV), and porcine chexenvirus-1 (PTV-1); and cardiogenic viruses, such as Theylvirus and encephalomyocarditis virus. 2A linkers derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively.

[0204] The P2A adapter may have a sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22. The T2A adapter may have a sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0205] Various adapter sequences are known in the art, including but not limited to glycine-serine (GS) spacers (also known as GS adapters), such as (GS)n, (SG)n, (GSGGS)n, and (GGGS)n, where n represents an integer of at least 1. Exemplary adapter sequences may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 25.

[0206] The nucleic acids disclosed herein may contain restriction enzyme site sequences.

[0207] The nucleic acids disclosed herein can be operatively linked to transcriptional control elements, such as promoters and enhancers.

[0208] The promoter can be a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK cell-specific promoter. For example, the CD4 gene promoter can be used; see, for example, Salmon et al., Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al., (2003) Blood 101:3416. As another example, the CD8 gene promoter can be used. NK cell-specific expression can be achieved by using the NcrI (p46) promoter; see, for example, Eckelhart et al., Blood (2011) 117:1565.

[0209] Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any nucleic acid sequence operatively linked to it. Other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, the Raoult sarcoma virus promoter, the EF-1α promoter, and human gene promoters (such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter). Furthermore, this disclosure is not limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of this disclosure. The use of inducible promoters provides a molecular switch capable of turning on the expression of a nucleic acid sequence operatively linked to it when such expression is desired, or turning off such expression when it is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0210] The nucleic acids disclosed herein may be provided for the production of (i) the Nef protein and / or Nef fusion protein described herein, and / or (ii) the CAR described herein (e.g., in mammalian cells). The nucleic acids disclosed herein may be provided for the amplification of such nucleic acids.

[0211] Nucleic acids described herein can be introduced into immune cells (e.g., T cells) or their precursors using vectors (e.g., expression vectors, such as lentiviral vectors). Vectors disclosed herein (e.g., lentiviral vectors) may contain one or more nucleic acids encoding the Nef protein and / or Nef fusion protein described herein. Vectors (e.g., lentiviral vectors) may contain additional elements that contribute to the functional expression of the Nef protein and / or Nef fusion protein and / or the CAR described herein. Expression vectors may contain mammalian promoters. Vectors may contain elongation factor-1-α promoters (EF-1α promoters). Using EF-1α promoters can increase the expression efficiency of downstream transgenes (e.g., nucleic acids encoding CARs). Physiological promoters (e.g., EF-1α promoters) are unlikely to induce integration-mediated genotoxicity and may eliminate the ability of retroviral vectors to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) may be incorporated into the vectors disclosed herein. Vectors (e.g., lentiviral vectors) may contain non-essential cis-acting sequences that can improve titers and gene expression.

[0212] Nucleic acids can encode naked CARs. The nucleic acid may contain, from the 5' to the 3' end, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB co-stimulatory signal transduction domain, and a primary intracellular signal transduction domain. In some cases, the nucleic acid encodes an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14.

[0213] Nucleic acids can encode (1) a CAR and (2) a Nef protein or a Nef fusion protein. Nucleic acids may contain the coding sequence for the Nef protein or Nef fusion protein, a 2A cleavable adapter, and the CAR from the 5' to the 3' end.

[0214] Nucleic acids can also encode protein biomarkers. These protein biomarkers can be dihydrofolate reductase (DHFR) proteins (e.g., mutant DHFR proteins). Mutant DHFR proteins can have an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20.

[0215] Nucleic acids can also encode exogenous cytokines or fragments thereof. Exogenous cytokines or fragments thereof can be membrane-bound interleukin 12β subunit (IL12p40) protein. The IL12p40 protein can have an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21.

[0216] Nucleic acid sequences encoding Nef proteins, Nef fusion proteins, engineered receptors, protein markers, and exogenous cytokines or fragments thereof can be linked via one or more nucleic acid sequences encoding cleavable adapters. Cleavable adapters can be P2A adapters or T2A adapters.

[0217] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). The length of the reference sequence aligned for comparison purposes is at least 80% of the reference sequence length, and may be at least 90%, 95%, or 100%. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of identical positions shared by the two sequences, taking into account the number of vacancies that need to be introduced to achieve optimal alignment and the length of each vacancies. For example, a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5 can be used to perform sequence comparison and determine the percentage of identity between two sequences.

[0218] Treatment

[0219] Engineered cells with reduced endogenous TCR levels (e.g., engineered immune cells), particularly those overexpressing the Nef protein and / or Nef fusion protein described herein, can be used in a variety of experimental, therapeutic, and commercial applications.

[0220] In one respect, this disclosure provides a pharmaceutical composition comprising the engineered cells and pharmaceutically acceptable carriers described herein.

[0221] In one aspect, this disclosure provides a method for treating a disease or disorder in a subject (e.g., a human subject), the method comprising administering to the subject an effective amount of the engineered cells or pharmaceutical composition described herein. The disease or disorder may be cancer, an autoimmune disease, or an infection.

[0222] Diseases or disorders can be solid tumors. A "solid tumor" is an abnormal mass of tissue that does not typically contain cysts or fluid-filled areas. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Different types of solid tumors are named after the types of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemia (blood cancers) typically does not form solid tumors.

[0223] In one respect, this disclosure provides a method for modulating an immune response, the method comprising administering an effective amount of the engineered cells described herein to a subject in need.

[0224] As used herein, the term "effective dose" means the amount that is effective at the necessary dosage and duration to achieve the desired result.

[0225] On the other hand, this disclosure provides a method for treating cancer, comprising administering an effective amount of the engineered cells described herein to a subject in need. Examples of cancers that can be treated include, but are not limited to, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNC), neuroendocrine prostate cancer (NEPC), pancreatic neuroendocrine tumor (PNET), gastrointestinal neuroendocrine carcinoma, leukemia (including chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and T-cell and B-cell leukemia), lymphoma (Hodgkin's and non-Hodgkin's), lymphoproliferative disorders, plasmacytoma, histiocytoma, melanoma, adenoma, sarcoma, solid tissue cancer, hypoxic tumors, squamous cell carcinoma, genitourinary cancers (such as cervical cancer and bladder cancer), hematopoietic system cancers, head and neck cancers, and nervous system cancers.

[0226] This disclosure further includes the use of the engineered cells described herein in the manufacture of medicaments or pharmaceutical compositions for modulating immune responses, treating infections, or treating cancers as described above.

[0227] Engineered cells can also be used in experimental models, for example, to further study and elucidate cell function.

[0228] One or more of the engineered cells described herein can be administered to a subject in a single, uniform form (such as intravenous injection) or in multiple forms (e.g., as multiple intravenous infusions or injections, or subcutaneous injections). In some cases, the engineered cells can be expanded in the subject after administration. The engineered cells can be frozen to provide cells for multiple treatments with the same cell preparation. The engineered cells disclosed herein and pharmaceutical compositions containing the engineered cells disclosed herein can be packaged as kits. Kits may include instructions for use of the engineered cells and compositions containing the engineered cells (e.g., written instructions).

[0229] Treatment methods may include administering a therapeutically effective amount of engineered cells to the subject. The therapeutically effective amount of engineered cells may be administered for at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. The therapeutically effective amount of engineered cells may be administered for at least one week. The therapeutically effective amount of engineered cells may be administered for at least two weeks.

[0230] The engineered cells described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administration can vary. For example, engineered cells can be used as a preventative agent and can be administered continuously to subjects with a condition or predisposition to reduce the likelihood of developing the disease or condition. Engineered cells can be administered to subjects during or as soon as possible after the onset of symptoms. Administration of engineered cells can begin immediately upon the onset of symptoms, within 3 hours before the onset of symptoms, within 6 hours before the onset of symptoms, within 24 hours before the onset of symptoms, within 48 hours before the onset of symptoms, or at any time after the onset of symptoms. Initial administration can be via any practical route (e.g., intravenous infusion or injection), such as any of the routes described herein using any of the formulations described herein. In some instances, administration of the engineered cells disclosed herein is intravenous. After the onset of cancer or an infectious disease, one or more doses of engineered cells can be administered as soon as practicable, and the duration of treatment can be as long as necessary, such as approximately 24 hours to approximately 48 hours, approximately 48 hours to approximately 1 week, approximately 1 week to approximately 2 weeks, approximately 2 weeks to approximately 1 month, and approximately 1 month to approximately 3 months. For cancer treatment, one or more doses of engineered cells can be administered several years after the onset of cancer and before or after other treatments. In some instances, engineered cells can be administered for at least about 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. The duration of treatment can vary for each subject.

[0231] Methods for administering engineered cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, adoptive T-cell therapy methods are described in, for example, U.S. Patent Application Publication No. 2003 / 0170238 by Gruenberg et al.; U.S. Patent No. 4,690,915 by Rosenberg; Rosenberg (2011) Nat RevClin Oncol. 8(10): 577-85. See, for example, Themeli et al., (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al., (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al., (2013) PLoS ONE 8(4): e61338. Cell therapy (e.g., adoptive T-cell therapy) can be performed via autologous transfer, wherein cells are isolated from and / or otherwise prepared from a subject to receive cell therapy or from a sample derived from such a subject. Therefore, in some respects, the cells are derived from the subjects who require treatment (e.g., patients), and after separation and processing, the cells are administered to the same subjects.

[0232] Cell therapy (e.g., adoptive T-cell therapy) can be performed via allogeneic transfer, where cells are isolated and / or otherwise prepared from a subject other than the one to be or ultimately receive the cell therapy (e.g., a first subject). In such embodiments, the cells are then administered to a different subject of the same species (e.g., a second subject). The first and second subjects may be genetically identical. The first and second subjects may be genetically similar. The second subject may express the same HLA class or supertype as the first subject.

[0233] The subject (e.g., a human subject) may have been treated with a therapeutic agent targeting a disease or condition (e.g., a tumor) prior to administration of the cells or a composition containing cells. The subject may be refractory or unresponsive to other therapeutic agents. The subject may have a persistent or relapsing disease, for example, after treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. In some cases, administration may be effective in treating the subject even though they have become resistant to another therapy.

[0234] The subject may respond to another treatment agent, and treatment with that agent reduces the disease burden. The subject may initially respond to the treatment agent but exhibit a relapse of the disease or condition over time. The subject may not experience a relapse. The subject may be identified as being at risk of relapse, such as being at high risk of relapse, and therefore prophylactically administered cells, for example, to reduce the likelihood of relapse or to prevent relapse. The subject may not have previously received treatment with another treatment agent.

[0235] Subjects may have persistent or recurrent diseases, for example, after treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. Even if the subject has become resistant to another therapy, administration can still effectively treat the subject.

[0236] The engineered cells described in this article can be administered to animals (such as mammals, and even humans) to treat cancer. Additionally, engineered cells can be used to treat any condition associated with cancer, particularly cell-mediated immune responses against one or more tumor cells, where treatment or mitigation of the disease is desired.

[0237] The engineered cells described herein (e.g., immune cells, T cells, or NK cells) may be included in a composition for immunotherapy. The composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition containing engineered cells may be administered.

[0238] The engineered cells can be used immediately for the aforementioned therapeutic, experimental, or commercial applications, or they can be cryopreserved for later use. The pharmaceutical composition may be included in a container, package, or dispenser along with the instructions for use.

[0239] The engineered cells disclosed herein can be formulated into unit dosage forms suitable for precise single-dose administration. The unit dosage form may contain additional lymphocytes. In the unit dosage form, the formulation is divided into unit doses containing appropriate amounts of one or more compounds. The unit doses may be in the form of packages containing discrete amounts of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions may be packaged in single-dose, non-resealable containers. Multi-dose, resealable containers may be used, for example, with or without preservatives. Pharmaceutical compositions may not contain preservatives. Formulations for parenteral injection may be presented in unit dosage forms, such as in ampoules or in multi-dose containers with preservatives.

[0240] Example

[0241] The disclosure is further described in the following examples, which do not limit the scope of the disclosure as set forth in the claims.

[0242] Example 1. Regulation of TCRαβ and CD3ζ protein expression by truncating SIV Nef M116

[0243] As an exemplary Nef protein in this disclosure, SIV Nef M116 is a mutant SIV Nef protein possessing a "dileucine-based AP recruitment domain" of mutant SIV Nef. The amino acid sequence of the wild-type SIV Nef protein is shown in SEQ ID NO: 1. M116 has two amino acid mutations relative to the wild-type SIV Nef protein: L178A and M179A. SIV Nef M116 can effectively downregulate the expression of the TCR complex on the surface of T cells, while having negligible downregulation of CD4 and CD28 expression. Details of SIV Nef M116 (amino acids 1-223, SEQ ID NO: 2) can be found, for example, in PCT application publication number WO2020020359 (which is incorporated herein by reference in its entirety). The full-length SIV Nef M116 protein was truncated to obtain SIV NefM116 Trun1 (supplementary amino acids 50-91 of SIV Nef M116; SEQ ID NO: 3), SIV Nef M116 Trun2 (supplementary amino acids 207-223 of SIV Nef M116; SEQ ID NO: 4) and SIV Nef M116 Trun3 (supplementary amino acids 50-91 and 207-223 of SIV Nef M116; SEQ ID NO: 5).

[0244] 1) Construction of expression plasmid encoding truncated SIV Nef M116

[0245] The pLVX-Puro plasmid (purchased from Clontech) was digested with ClaI and EcoRI restriction enzymes to remove the CMV promoter, and the human EF1α promoter (GenBank: J04617.1) was cloned into the digested plasmid to obtain the pLVX-hEF1α plasmid.

[0246] The nucleic acid sequence encoding a truncated SIV Nef M116 was cloned downstream of the human EF1α promoter on the expression plasmid pLVX-hEF1α. The truncated SIV Nef M116 was ligated to a selection marker protein (Puro; SEQ ID NO: 19) via the self-cleaving peptide T2A (SEQ ID NO: 22). The corresponding recombinant expression plasmids PLLV-modified 116 (“M116”, encoding SIVNef M116), PLLV-modified 708 (“M708”, encoding SIV Nef M116 Trun1), PLLV-modified 2237 (“M2237”, encoding SIV Nef M116 Trun2), and PLLV-modified 2729 (“M2729”, encoding SIV Nef M116 Trun3) were isolated and mixed with helper plasmids (e.g., psPAX2 and pMD2.G) for co-transfection into HEK 293T cells to generate lentivirus. Sixty hours after transfection, the cell culture supernatant containing lentivirus was collected and centrifuged at 3000 rpm for 5 minutes at 4°C. The supernatant was then filtered through a 0.45 μm filter and further concentrated using a 500 KD hollow fiber membrane column tangential flow technique to prepare a lentivirus concentrate, which was stored at -80°C for later use.

[0247] 2) Preparation of Jurkat cell lines expressing truncated SIV Nef M116

[0248] Jurkat cells (human T lymphocytes, clone E6-1, ATCC, TIB-152™) were cultured in 90% RPMI 1640 medium (Gibco, catalog number: 22400-089) supplemented with 10% fetal bovine serum (FBS, Gibco, catalog number: 10099-141C). Five cells were infected with the lentiviruses prepared above. 6 Jurkat cells were collected, and the cell suspension was added to 10 cm culture dishes, which were then incubated at 37°C with 5% CO2 for 3 days. On the third day of virus transduction, the culture medium was replaced to include 1 μg / mL puromycin (Gibco, catalog number: A1113803). Culture was continued for another three days for resistance selection.

[0249] 3) Detection of TCRαβ and CD3ζ expression by flow cytometry

[0250] The expression of TCRαβ was detected by flow cytometry as follows. 3 × 10⁻⁶ cells were used. 6The cell suspension was centrifuged at 1000 rpm for 1 minute at room temperature, and the supernatant was discarded. The cells were resuspended in DPBS (Cytiva, catalog number: SH30028.02) and incubated with 1 μL (1:100) APC anti-human TCRα / β antibody (Miltenyi, catalog number: 130-113-527) at 4°C for 30 minutes. After incubation, the cell suspension was centrifuged at 1000 rpm for 1 minute at room temperature. The supernatant was discarded, and the cells were resuspended in 1 mL of DPBS. The cells were washed twice as described above, and then analyzed by flow cytometry.

[0251] The expression of CD3ζ was detected by flow cytometry as follows. The cell suspension was centrifuged at 1000 rpm for 1 minute at room temperature, and the supernatant was discarded. 200 μL of BD Cytofix™ fixation buffer (BD Biosciences, catalog number: 554655) was added to the cell sample. The cells were resuspended and incubated at room temperature for 10 minutes. The cell suspension was centrifuged at 300 g for 1 minute at room temperature, and the supernatant was discarded. The cells were resuspended with 200 μL of BD Perm / Wash™ buffer (BD Biosciences, catalog number: 51-2091KZ) and washed once. Then, 1 μL (1:100) of PE anti-CD247 (TCRζ, CD3ζ) antibody (BioLegend, catalog number: 644106) was added, and the cells were incubated at room temperature for 30 minutes. The cells were washed twice as described above, and then flow cytometry was performed.

[0252] Figure 1A The expression of TCRαβ in mimicry, M116-Jurkat, M708-Jurkat, M2237-Jurkat, and M2729-Jurkat cells was shown, with the percentages of TCRαβ+ cells being 94.96%, 19.86%, 38.36%, 14.28%, and 38.04%, respectively. Compared to the blank mimicry group (untransduced Jurkat cells), TCRαβ expression was significantly downregulated in Jurkat cells transduced with plasmids M116, M708, M2237, and M2729, respectively.

[0253] Figure 1B CD3ζ expression was shown in mimicry, M116-Jurkat, M708-Jurkat, M2237-Jurkat, and M2729-Jurkat cells, with the percentages of CD3ζ+ cells being 99.72%, 98.68%, 99.05%, 98.70%, and 98.77%, respectively. CD3ζ expression was not regulated.

[0254] The results indicate that, similar to SIV Nef M116, truncated SIV Nef M116 (SIV Nef M116 Trun1, SIV Nef M116 Trun2 and SIV Nef M116 Trun3) can significantly downregulate TCRαβ on T lymphocytes without degrading CD3ζ protein.

[0255] Example 2. Regulation of TCRαβ and CD3ζ protein expression in CAR-T cells using a truncated SIV Nef M116 and TRIM21 Trun2 fusion protein.

[0256] 1) Construction of expression plasmids

[0257] To further degrade endogenous CD3ζ protein in T lymphocytes, a fusion protein of truncated SIV Nef and E3 ubiquitin ligase (e.g., TRIM21) was constructed and expressed in CAR-T cells. The TRIM21 protein containing a RING-Box coiled-coil domain (RBCC) (amino acids 1-267 of TRIM21; SEQ ID NO: 11) was truncated to obtain TRIM21 Trun2 (amino acids 1-85 of TRIM21; SEQ ID NO: 12). In some embodiments, a linker connecting the truncated SIV Nef to TRIM21 Trun2 is present. An exemplary linker has the amino acid sequence shown in SEQ ID NO: 25.

[0258] Similar to the method described in Example 1, the DNA fragment encoding SIV Nef M116-linker-TRIM21 Trun2-T2A-CD8αSP-CD20 scFv(Leu16)-CD8α hinge-CD8α TM-4-1BB-ITAM010-P2A-DHFR L22F / F31S-P2A-SP-MB12 was cloned into the expression plasmid pLVX-hEF1α to obtain the expression plasmid PLSINK modified 2268 (referred to as "M2268"). The DNA fragment encoding the truncated SIV Nef M116-linker-TRIM21 Trun2-T2A-CD8α SP-CD20 scFv(Leu16)-CD8α hinge-CD8α TM-4-1BB-ITAM010-P2A-DHFR L22F / F31S-P2A-SP-MB12 was cloned into the expression plasmid pLVX-hEF1α to obtain the PLSINK-modified plasmids 2410 (SIV Nef M116Trun1, abbreviated as "M2410"), 2445 (SIV Nef M116 Trun2, abbreviated as "M2445"), and 2444 (SIV Nef M116 Trun3, abbreviated as "M2444"), respectively. Schematic structures and sequences of the fusion proteins are shown in Table 1.

[0259] Table 1. Exemplary fusion proteins with truncated SIV Nef M116 and TRIM21 Trun2 in the corresponding CD20 CAR expression structures.

[0260]

[0261] Lentiviral viruses were generated and concentrated using a method similar to that described in Example 1. Recombinant expression plasmids were isolated and mixed with helper plasmids (e.g., pMDLg-pRRE, pRSV-Rev, and pMD2.G) for co-transfection of HEK 293T cells to generate lentivirus. In some embodiments, M2410, M2445, and M2444, having truncated SIV NefM116, could increase viral titers by 1.53 to 1.78 times, 2.50 to 2.59 times, and 5.82 to 8.05 times, respectively, compared to M2268 having the full-length SIV Nef M116.

[0262] The CD20 CAR modified with ITAM010 (CD8α SP-CD20 scFv (Leu16)-CD8α hinge-CD8α TM-4-1BB-ITAM010) is disclosed in PCT application publication number WO 2021037221, and its amino acid sequence is shown in SEQ ID NO: 13. ITAM010 is disclosed in PCT application publication number WO 2021037221, and its sequence is shown in SEQ ID NO: 15. The amino acid sequence of dihydrofolate reductase (DHFR) L22F / F31S is shown in SEQ ID NO: 20. MB12 is an exemplary membrane-binding IL12p40 polypeptide having a schematic structure of IL12p40-CD8α hinge-CD8α transmembrane domain-CD8α intracellular cytoplasmic domain, and its amino acid sequence is shown in SEQ ID NO: 21. The amino acid sequences of the self-cleaving peptides T2A, P2A, and signal peptide (SP) are shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively.

[0263] Jurkat cell lines were prepared according to the method described in Example 1 to obtain M2268-Jurkat, M2410-Jurkat, M22445-Jurkat, and M2444-Jurkat cells, respectively. No puromycin resistance screening was performed on the cultures.

[0264] 2) Detection of CAR, TCRαβ and CD3ζ expression by flow cytometry

[0265] CAR expression was detected using the flow cytometry method for TCRαβ described in Example 1. Specifically, cell staining was performed using 0.64 μL Alexa Flour™ 7488 LUCAR-20S ADA antibody (manufactured by GeneScript, order number: LGBUADAb-1), 1 μL (1:100) APC anti-human TCRα / β antibody, and 1 μL (1:100) PE anti-CD247 (TCRζ, CD3ζ) antibody (BioLegend, catalog number: 644106).

[0266] Figure 2AThe expression of TCRαβ in mimicry, M2268-Jurkat, M2410-Jurkat, M2445-Jurkat and M2444-Jurkat cells was shown, and the percentages of TCRαβ-positive (TCRαβ+) cells in CAR-positive (CAR+) cells were 97.57%, 3.85%, 3.04%, 4.17% and 3.36%, respectively.

[0267] Figure 2B The expression of CD3ζ in mimicry, M2268-Jurkat, M2410-Jurkat, M2445-Jurkat and M2444-Jurkat cells was shown, and the percentage of CD3ζ+ cells in CAR+ cells was 97.87%, 16.63%, 15.46%, 16.11% and 12.51%, respectively.

[0268] Compared with the blank mimic group (untransduced Jurkat cells), the expression of TCRαβ and CD3ζ was significantly downregulated in Jurkat cells transduced with plasmids M2268, M2410, M2445 and M2444, respectively.

[0269] The results indicate that the SIV Nef M116-TRIM21 Trun2, SIV Nef M116 Trun1-TRIM21 Trun2, SIV Nef M116 Trun2-TRIM21 Trun2 and SIV Nef M116 Trun3-TRIM21 Trun2 fusion proteins can significantly downregulate TCRαβ on T lymphocytes and capture and significantly degrade CD3ζ protein.

[0270] Example 3. Cell phenotype, CD3ζ protein expression, and specific cytotoxicity of CD20 CAR-T cells containing a fusion protein of truncated SIV Nef M116 and TRIM21 Trun2.

[0271] 1) Construction of expression plasmids

[0272] The expression plasmid was constructed using the method described in Example 2. A DNA fragment encoding SIV Nef M116 Trun3-TRIM21Trun2-T2A-CD8α SP-CD20 scFv (3H7)-CD8α hinge-CD8α TM-4-1BB-ITAM045-P2A-DHFRL22F / F31S-P2A-SP-MB12 was cloned into the expression plasmid pLVX-hEF1α to obtain the PLSINK-modified expression plasmid 2664 (referred to as "M2664"). Lentivirality was generated and concentrated as described in Example 2.

[0273] The amino acid sequence of CD20 scFv (3H7) is shown in SEQ ID NO: 18. ITAM045 is disclosed in PCT application publication number WO 2021037221, and its sequence is shown in SEQ ID NO: 16. The amino acid sequence of the ITAM045-modified CD20 CAR (CD8α SP-CD20 scFv (3H7)-CD8α hinge-CD8α TM-4-1BB-ITAM045) is shown in SEQ ID NO: 14. The amino acid sequence of the fusion protein of SIV Nef M116 Trun3 and TRIM21 Trun2 is shown in SEQ ID NO: 10.

[0274] 2) T cell isolation, transduction, and preparation

[0275] 50 mL of fresh peripheral blood was collected from volunteers, and peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte separation medium and density gradient centrifugation. T lymphocytes were isolated and purified by labeling cells with magnetic beads using the Pan T Cell Isolation Kit (Miltenyi, catalog number: 130-096-535). The purified T cells were treated with CD3 / CD28 magnetic beads for T lymphocyte activation and proliferation. After culturing at 37°C in a 5% CO2 incubator for 24 hours, 5 × 10⁶ cells were separated and centrifuged. 6 Activated T lymphocytes were infected with the M2664 lentiviral vector. Cell suspensions were added to 6-well plates and cultured at 37°C in a 5% CO2 incubator. The CD3ζ gene (CD247) was knocked out in UnT (untransduced T cells) using CRISPR / Cas9 technology to generate CD3ζ KO-T cells. On day 17 of viral transduction, TCRαβ-negative cells were enriched and sorted using a TCRα / β sorting kit to complete the preparation of CD3ζ KO-T and M2264-T cells.

[0276] 3) Detection of CD5, CAR, TCRαβ, MB12 and CD3ζ expression by flow cytometry

[0277] The expression of CD5, CAR, TCRαβ, and MB12 was detected using the flow cytometry method for TCRαβ as described in Example 1. Specifically, cell staining was performed using 1 μL (1:100) PE / cyanin 7 anti-human CD5 antibody, 0.64 μL Alexa Flour™ 7488 LUCAR-20S ADA antibody (manufactured by GeneScript, order number: LGBUADAb-1), 1 μL (1:100) APC anti-human TCRα / β antibody, and 1 μL (1:100) PE anti-human IL-12 / IL-23 p40 antibody.

[0278] The expression of CD3ζ is detected according to the method described in Example 1.

[0279] 4) Detection of CD3ζ by Western blot

[0280] 5 × 10 6 UnT, CD3ζ KO-T, and 2664-T cells were washed twice with cold DPBS and then centrifuged at 2500 g for 5 minutes. After centrifugation, cells were collected and 1 mL of cell lysis buffer (Thermo Scientific, catalog number: 89900) was added. The cell lysate was gently vortexed on ice for 15 minutes and then centrifuged at 14,000 g for 15 minutes at 4°C. The supernatant was transferred to new centrifuge tubes and stored at -80°C for later use. The expression level of CD3ζ protein was detected by Western blotting using a CD3ζ-specific antibody (Santa Cruz, catalog number: sc-1239), and β-actin was detected (as a control).

[0281] 5) Specific cytotoxicity of CAR-T cells

[0282] In Corning ® In 384-well plates (white), each T cell group was mixed with Raji.Luc (CD20-positive lymphoma cells, ATCC, catalog number: CCL-86) cells at effector-to-target cell ratios of 20:1 and 10:1, and incubated for 18–24 hours. The ONE-Glo™ luciferase assay system (TAKARA, catalog number: B6120) was used.

[0283] Specifically, 25 μL of One-Glo™ reagent was added to each well of a 384-well plate and analyzed using a microplate reader (TECAN, Spark). ® 10M) Detected the fluorescence signal from luciferase. The killing efficiency of each group of lymphocytes against target cells was calculated as follows: Killing efficiency (%) = (1 - (RLU experimental group - RLU blank control group) / RLU tumor cell group) × 100%.

[0284] Figure 3A and Figure 3B The results showed that after TCRαβ sorting and enrichment of M2664-T cells, the percentage of TCRαβ-CAR+ (TCRαβ negative and CAR positive) cells was 83.20%, and the percentage of CAR+MB12+ cells was 79.05%.

[0285] Figure 4AThe expression of CD3ζ in UnT, CD3ζ KO-T, and M2664-T cells was shown by flow cytometry, and the percentages of CD3ζ+ cells were 99.10%, 0.21%, and 1.55%, respectively.

[0286] Figure 4B The expression of CD3ζ protein in UnT, CD3ζ KO-T, and M2664-T cells was shown by Western blot analysis. Compared with UnT, CD3ζ expression was significantly downregulated in CD3ζ KO-T and M2664-T cells.

[0287] The results indicate that the fusion protein of SIV Nef M116 Trun3 and TRIM21 Trun2 on CAR-T cells has the function of capturing and degrading CD3ζ protein. Furthermore, CAR-T cells expressing the fusion protein of SIV Nef M116 Trun3 and TRIM21 Trun2 exhibited specific cytotoxicity. Figure 5 This has been verified.

[0288] Other embodiments

[0289] It should be understood that although this disclosure has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of this disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A non-naturally occurring negative regulatory factor (Nef) protein, said non-naturally occurring Nef protein comprising the deletion of amino acids 207-223 or the corresponding amino acids numbered as in SEQ ID NO: 1 and SEQ ID NO:

2.

2. The non-naturally occurring Nef protein as described in claim 1, wherein the non-naturally occurring Nef protein comprises amino acids 50-91 numbered as in SEQ ID NO: 1 and SEQ ID NO: 2, or the deletion of the corresponding amino acids thereof.

3. The non-naturally occurring Nef protein as described in claim 1 or claim 2, wherein the non-naturally occurring Nef protein downregulates the cell surface expression of the endogenous T cell receptor (TCR).

4. The non-naturally occurring Nef protein according to any one of claims 1-3, wherein the non-naturally occurring Nef protein consists of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO:

5.

5. A fusion protein comprising (1) a non-naturally occurring Nef protein and (2) an E3 ubiquitin ligase or a fragment thereof.

6. The fusion protein of claim 5, wherein the non-naturally occurring Nef protein comprises the deletion of amino acids 207-223 or the corresponding amino acids numbered in SEQ ID NO:1 and SEQ ID NO:

2.

7. The fusion protein of claim 5 or claim 6, wherein the non-naturally occurring Nef protein comprises amino acids 50-91 numbered as in SEQ ID NO: 1, SEQ ID NO: 2, or the deletion of the corresponding amino acids thereof.

8. The fusion protein according to any one of claims 5-7, wherein the non-naturally occurring Nef protein consists of an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

5.

9. The fusion protein according to any one of claims 5-8, wherein the E3 ubiquitin ligase or a fragment thereof is derived from the triple motif protein 21 (TRIM21).

10. The fusion protein of claim 9, wherein the TRIM21 protein is truncated; optionally, wherein the TRIM21 protein comprises a RING domain.

11. The fusion protein of claim 9 or claim 10, wherein the TRIM21 protein comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12; optionally, wherein the amino acid sequence of the TRIM21 protein is shown in SEQ ID NO:

12.

12. The fusion protein of any one of claims 5-11, wherein the non-naturally occurring Nef protein is linked to the E3 ubiquitin ligase or a fragment thereof via a linker; optionally wherein the linker comprises the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO:

25.

13. The fusion protein according to any one of claims 5-11, wherein the non-naturally occurring Nef protein is directly linked to the E3 ubiquitin ligase or a fragment thereof without a linker.

14. The fusion protein according to any one of claims 5-13, wherein the non-naturally occurring Nef protein is located at the N-terminus of the E3 ubiquitin ligase or a fragment thereof, or at the C-terminus of the E3 ubiquitin ligase or a fragment thereof.

15. The fusion protein according to any one of claims 5-14, wherein the fusion protein comprises an amino acid sequence of any one of SEQ ID NO: 6-10 or an amino acid sequence having at least 90%, 95% or 99% identity with an amino acid sequence of any one of SEQ ID NO: 6-10.

16. The fusion protein of claim 15, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO:

10.

17. The fusion protein according to any one of claims 5-16, wherein the fusion protein downregulates and / or degrades endogenous CD3ζ.

18. A nucleic acid comprising a first nucleic acid sequence encoding a non-naturally occurring Nef protein as described in any one of claims 1-4 or a fusion protein as described in any one of claims 5-17.

19. The nucleic acid of claim 18, wherein the nucleic acid further comprises a second nucleic acid sequence encoding an engineered receptor.

20. The nucleic acid of claim 19, wherein the engineered receptor is a chimeric antigen receptor (CAR); optionally wherein the CAR comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO:

14.

21. The nucleic acid of any one of claims 18-20, wherein the nucleic acid further comprises a nucleic acid sequence encoding a protein marker; optionally wherein the protein marker is a mutant dihydrofolate reductase (DHFR) polypeptide having the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO:

20.

22. The nucleic acid of any one of claims 18-21, wherein the nucleic acid further comprises a nucleic acid sequence encoding a foreign cytokine or a fragment thereof; optionally wherein the foreign cytokine or fragment thereof is a membrane-bound interleukin 12β subunit (IL12p40) polypeptide having the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO:

21.

23. The nucleic acid of any one of claims 19-22, wherein each nucleic acid sequence is linked via a nucleic acid sequence encoding a cleavable adapter; optionally, wherein the cleavable adapter is P2A or T2A.

24. A vector comprising the nucleic acid as described in any one of claims 18-23.

25. An engineered cell comprising a non-naturally occurring Nef protein as described in any one of claims 1-4, a fusion protein as described in any one of claims 5-17, a nucleic acid as described in any one of claims 18-23, or a vector as described in claim 24.

26. The engineered cell of claim 25, wherein the engineered cell further comprises an engineered receptor; optionally wherein the engineered receptor is selected from the group consisting of engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), and T-cell antigen conjugates (TACs).

27. The engineered cell of claim 26, wherein the engineered receptor comprises: (a) Extracellular ligand-binding domain; (b) Transmembrane domains; and (c) Intracellular signal transduction domain (ISD), the ISD comprising a chimeric signal transduction domain (CMSD) having an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO:

16.

28. The engineered cell of claim 27, wherein the engineered receptor further comprises an N-terminus signal peptide located in the extracellular ligand binding domain.

29. The engineered cell according to any one of claims 26-28, wherein the extracellular ligand-binding domain of the engineered receptor binds to the antigen. The antigens mentioned therein are selected from the group consisting of: BCMA, CLL1, CD4, GPC3, GPRC5D, GU2CYC, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MAGEA3, p53, MART1, GP100, protease-3 (PR3), tyrosinase, survival protein, hTERT, EphA2, NY-ESO-1, h5T4, PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, duracin 18.2, duracin 6, NKG2D, delta-like ligand 3 (DLL3), CD70, ADGRE2, FcRH5, NKp80, NKp30, NKG2A, CD229, and CS-1.

30. The engineered cell according to any one of claims 26-29, wherein the engineered receptor is a CAR; optionally wherein: The intracellular signal transduction domain of the CAR further includes a co-stimulatory signal transduction domain, and the CAR further includes a hinge domain located between the C-terminus of the extracellular ligand binding domain and the N-terminus of the transmembrane domain.

31. The engineered cell according to any one of claims 25-30, wherein the engineered cell comprises a CAR, the CAR comprising an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO:

14.

32. The engineered cell according to any one of claims 25-31, wherein the engineered cell comprises a protein marker; optionally, the protein marker is a mutant DHFR polypeptide having the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO:

20.

33. The engineered cell according to any one of claims 25-32, wherein the engineered cell comprises an exogenous cytokine or a fragment thereof; optionally wherein the exogenous cytokine or fragment thereof is a membrane-bound IL12p40 polypeptide having the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO:

21.

34. The engineered cell according to any one of claims 25-33, wherein the engineered cell is selected from the group consisting of: T cells, αβT cells, γδT cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells and embryonic stem cells.

35. The engineered cell of claim 34, wherein the cell is a T cell.

36. The engineered cell of claim 35, wherein the engineered cell expresses a reduced level of CD3ζ compared to the precursor T cell, and / or expresses a reduced level of endogenous T cell receptor (TCR) compared to the precursor T cell.

37. The engineered cells of claim 35 or 36, wherein the engineered cells do not elicit a graft-versus-host disease (GvHD) response or elicit a reduced GvHD response in tissue-incompatible individuals compared to a GvHD response elicited by primary T cells isolated from a donor from which the engineered cells are derived.

38. A method for producing engineered T cells, the method comprising introducing a nucleic acid as described in any one of claims 18-23 or a vector as described in claim 24 into a precursor cell; optionally, wherein the method further comprises introducing a second nucleic acid encoding a chimeric antigen receptor (CAR) into the precursor cell.

39. A pharmaceutical composition comprising engineered cells and a pharmaceutically acceptable carrier as described in any one of claims 25-38.

40. A method of treating a disease or disorder in a subject, the method comprising administering to a subject in need a therapeutically effective amount of engineered cells as described in any one of claims 25-38 or a pharmaceutical composition as described in claim 39.

41. A method for downregulating and / or degrading a target protein in T cells from a subject, the method comprising introducing a nucleic acid as described in any one of claims 18-23 or a vector as described in claim 24 into the T cells; optionally wherein the target protein is CD3ζ and / or TCR.

42. A chimeric antigen receptor (CAR), said CAR comprising: (1) An extracellular ligand-binding domain that binds to CD20, wherein the extracellular ligand-binding domain comprises: LCDR1 containing the amino acid sequence of SEQ ID NO: 26, LCDR2 containing the amino acid sequence of SEQ ID NO: 27, LCDR3 containing the amino acid sequence of SEQ ID NO: 28, HCDR1 containing the amino acid sequence of SEQ ID NO: 29, HCDR2 containing the amino acid sequence of SEQ ID NO: 30, and HCDR3 containing the amino acid sequence of SEQ ID NO: 31; wherein the CDR is determined according to the Kabat numbering scheme. (2) Transmembrane domains, and (3) Intracellular signal transduction domain (ISD), wherein the ISD comprises a chimeric signal transduction domain (CMSD) having an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO: 16; optionally wherein the intracellular signal transduction domain further comprises a co-stimulatory signal transduction domain.

43. The CAR of claim 42, wherein the extracellular ligand binding domain of the CAR comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO:

18.

44. The CAR of claim 42 or claim 43, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence of SEQ ID NO: 14.

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