Enhanced chimeric antigen receptor and application thereof

Abstract

The invention provides an enhanced chimeric antigen receptor which comprises an extracellular antigen recognition structural domain, a hinge region, a transmembrane region and an intracellular structural domain, the intracellular structural domain comprises a costimulatory signal transduction region and an intracellular signal transduction region which are sequentially connected from an N terminal to a C terminal, and the intracellular structural domain further comprises an amino acid sequence derived from CD7. Compared with a CAR-T cell with a conventional second-generation chimeric antigen receptor structure, the CAR-T cell with the enhanced chimeric antigen receptor structure has better continuous amplification capability and continuous tumor killing capability.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to an enhanced chimeric antigen receptor and its applications. Background Technology

[0002] Chimeric antigen receptors (CARs) are the core components of CAR cell therapy drugs, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. CAR-T cell immunotherapy is considered one of the most promising methods for conquering tumors. CAR-T cells utilize genetic modification to express CAR proteins on the surface of T cells. These CAR proteins are capable of recognizing intact proteins on the surface of tumor cell membranes without relying on antigen presentation, thereby activating and functionally affecting T cells.

[0003] In vivo, antigens expressed on the surface of tumor cells can activate CAR-T cells and cause them to proliferate. However, continuous activation of T cells leads to cell exhaustion. Exhausted T cells will have reduced proliferative capacity and effector function. Therefore, improving the persistence of CAR-T cells determines the persistence of their function. Summary of the Invention

[0004] This application provides an enhanced chimeric antigen receptor and its application. The inventors verified and screened six candidate enhanced chimeric antigen receptor structures on T cells. Compared with CAR-T cells with conventional second-generation chimeric antigen receptor structures, CAR-T cells with the enhanced chimeric antigen receptor structure of this application have better sustained expansion and sustained tumor killing capabilities.

[0005] This application provides an enhanced chimeric antigen receptor comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. The intracellular domain comprises a co-stimulatory signal transduction region and an intracellular signal transduction region, wherein the intracellular domain further comprises an amino acid sequence derived from the CD7 protein.

[0006] In some embodiments of the aforementioned enhanced chimeric antigen receptor, the amino acid sequence derived from the CD7 protein is an amino acid sequence derived from the intracellular region of the CD7 protein.

[0007] In some embodiments of the enhanced chimeric antigen receptor described above, the amino acid sequence derived from the intracellular region of the CD7 protein comprises the amino acid sequence shown in SEQ ID NO:16; alternatively, the amino acid sequence derived from the intracellular region of the CD7 protein is shown in SEQ ID NO:16.

[0008] In some embodiments of the aforementioned enhanced chimeric antigen receptor, an amino acid sequence derived from the CD7 protein is linked between the transmembrane region and the co-stimulatory signal transduction region.

[0009] In some embodiments of the enhanced chimeric antigen receptor described above, the co-stimulatory signal transduction region comprises an amino acid sequence derived from 4-1BB or CD28; optionally, the co-stimulatory signal transduction region comprises an amino acid sequence derived from 4-1BB; more preferably, the co-stimulatory signal transduction region is an amino acid sequence derived from 4-1BB.

[0010] And / or, wherein the intracellular signal transduction region comprises one or more amino acids derived from CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signal transduction region comprises an amino acid sequence derived from CD3ζ; further optionally, the intracellular signal transduction region is an amino acid sequence derived from CD3ζ.

[0011] In some embodiments of the enhanced chimeric antigen receptor described above, the amino acid sequence derived from 4-1BB includes the amino acid sequence shown in SEQ ID NO:13; alternatively, the amino acid sequence derived from 4-1BB is shown in SEQ ID NO:13.

[0012] In some embodiments of the enhanced chimeric antigen receptor described above, the amino acid sequence derived from CD3ζ includes the amino acid sequence shown in SEQ ID NO:15; alternatively, the amino acid sequence derived from CD3ζ is shown in SEQ ID NO:15.

[0013] In some embodiments of the enhanced chimeric antigen receptor described above, the intracellular domain comprises an amino acid sequence as shown in SEQ ID NO:21; alternatively, the amino acid sequence of the intracellular domain is as shown in SEQ ID NO:21.

[0014] In some embodiments of the aforementioned enhanced chimeric antigen receptor, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, CD8α, and DAP10; optionally, the amino acids in the hinge region are derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises, as shown in SEQ ID NO.

[0015] The amino acid sequence shown in NO:11; more preferably, the amino acid sequence of the hinge region is as shown in SEQ ID.

[0016] Shown in NO:11.

[0017] In some embodiments of the enhanced chimeric antigen receptor described above, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70, DAP10, 2B4, DNAM-1, and HVEM; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:12; even more optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:12.

[0018] In some embodiments of the enhanced chimeric antigen receptor described above, a guide peptide is further attached to the N-terminus of the extracellular antigen recognition domain; optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO:1; further optionally, the amino acid sequence of the guide peptide is shown in SEQ ID NO:1.

[0019] This application provides an enhanced chimeric antigen receptor targeting CD7, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain targeting CD7. The intracellular domain includes a co-stimulatory signal transduction region and an intracellular signal transduction region, wherein the intracellular domain further comprises an amino acid sequence derived from the CD7 protein.

[0020] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the amino acid sequence derived from the CD7 protein is an amino acid sequence derived from the intracellular region of the CD7 protein.

[0021] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the amino acid sequence derived from the intracellular region of the CD7 protein comprises the amino acid sequence shown in SEQ ID NO:16; alternatively, the amino acid sequence derived from the intracellular region of the CD7 protein is shown in SEQ ID NO:16.

[0022] In some embodiments, the aforementioned enhanced chimeric antigen receptor targeting CD7 has an amino acid sequence derived from the CD7 protein linked between the transmembrane region and the co-stimulatory signal transduction region.

[0023] In some embodiments of the aforementioned CD7-targeting enhanced chimeric antigen receptor, the co-stimulatory signal transduction region comprises an amino acid sequence derived from 4-1BB or CD28; optionally, the co-stimulatory signal transduction region comprises an amino acid sequence derived from 4-1BB; further optionally, the co-stimulatory signal transduction region is an amino acid sequence derived from 4-1BB.

[0024] And / or, wherein the intracellular signal transduction region comprises one or more amino acids derived from CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signal transduction region comprises an amino acid sequence derived from CD3ζ; further optionally, the intracellular signal transduction region is an amino acid sequence derived from CD3ζ.

[0025] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the amino acid sequence derived from 4-1BB includes the amino acid sequence shown in SEQ ID NO:13; alternatively, the amino acid sequence derived from 4-1BB is shown in SEQ ID NO:13.

[0026] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the amino acid sequence derived from CD3ζ includes the amino acid sequence shown in SEQ ID NO:15; alternatively, the amino acid sequence derived from CD3ζ is shown in SEQ ID NO:15.

[0027] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the intracellular domain comprises an amino acid sequence as shown in SEQ ID NO:21; alternatively, the amino acid sequence of the intracellular domain is as shown in SEQ ID NO:21.

[0028] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the extracellular antigen recognition domain targeting CD7 comprises a CD7 antibody heavy chain variable region and a CD7 antibody light chain variable region. The amino acid sequences of CDR1, CDR2, and CDR3 in the CD7 antibody heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CD7 antibody light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO:4. Optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the CD7 antibody heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CD7 antibody light chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:4. The amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the antibody light chain shown in NO:4.

[0029] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CD7 antibody heavy chain respectively comprise the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CD7 antibody light chain respectively comprise the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CD7 antibody heavy chain are as shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CD7 antibody light chain are as shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.

[0030] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the variable region of the CD7 antibody heavy chain contains the amino acid sequence shown in SEQ ID NO:3, and the variable region of the CD7 antibody light chain contains the amino acid sequence shown in SEQ ID NO:4; optionally, the amino acid sequence of the variable region of the CD7 antibody heavy chain is as shown in SEQ ID NO:3, and the amino acid sequence of the variable region of the CD7 antibody light chain is as shown in SEQ ID NO:4.

[0031] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the extracellular antigen recognition domain targeting CD7 comprises a humanized CD7 scFv antibody.

[0032] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the extracellular antigen recognition domain targeting CD7 comprises the amino acid sequence shown in SEQ ID NO:2; alternatively, the extracellular antigen recognition domain targeting CD7 is the amino acid sequence shown in SEQ ID NO:2.

[0033] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, CD8α, and DAP10; optionally, the amino acids in the hinge region are derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:11; even more optionally, the amino acid sequence of the hinge region is as shown in SEQ ID NO:11.

[0034] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70, DAP10, 2B4, DNAM-1, and HVEM; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:12; even more optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:12.

[0035] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, a guide peptide is further attached to the N-terminus of the extracellular antigen recognition domain; optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO:1; further optionally, the amino acid sequence of the guide peptide is shown in SEQ ID NO:1.

[0036] In some embodiments, the enhanced chimeric antigen receptor targeting CD7 described above has an amino acid sequence as shown in SEQ ID NO:1-SEQ ID NO:2-SEQ ID NO:11-SEQ ID NO:12-SEQ ID NO:21, with the amino acids linked sequentially from the N-terminus to the C-terminus; optionally, the sequential linking is a direct linking.

[0037] This application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the aforementioned enhanced chimeric antigen receptors targeting CD7.

[0038] This application also provides a vector containing the isolated nucleic acid molecules described above.

[0039] This application also provides an engineered immune effector cell comprising the above-described enhanced chimeric antigen receptor targeting CD7, the above-described isolated nucleic acid molecule, or the above-described carrier.

[0040] In some embodiments, the engineered immune effector cells described above are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0041] In some embodiments, the engineered immune effector cells described above are T lymphocytes; alternatively, the T lymphocytes are autologous T lymphocytes or allogeneic T lymphocytes.

[0042] In some embodiments, the allogeneic T lymphocytes described above comprise CD7 / TRAC double-negative cells obtained through gene editing; further, the allogeneic T lymphocytes comprise 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, or 99% or more of CD7 / TRAC double-negative cells obtained through gene editing.

[0043] In some embodiments, the engineered immune effector cells described above are αβT lymphocytes or γδT lymphocytes.

[0044] This application also provides a pharmaceutical composition comprising the above-described engineered immune effector cells and pharmaceutically acceptable excipients.

[0045] In some embodiments of the above-described pharmaceutical composition, pharmaceutically acceptable excipients include protective agents.

[0046] In some embodiments of the above-described pharmaceutical composition, pharmaceutically acceptable excipients include cell cryopreservation solutions.

[0047] In some embodiments, the pharmaceutical composition described above is an intravenous injection preparation.

[0048] Furthermore, this application also provides a method for preparing engineered immune effector cells that target CD7, comprising the following steps: introducing a nucleotide sequence encoding any of the aforementioned enhanced chimeric antigen receptors that target CD7 into the immune effector cells.

[0049] In some embodiments of the above method, the immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0050] In some embodiments of the above method, the immune effector cells are T lymphocytes; optionally, the T lymphocytes are autologous T lymphocytes or allogeneic T lymphocytes.

[0051] In some embodiments of the above method, the T lymphocytes are αβT lymphocytes or γδT lymphocytes.

[0052] In some embodiments of the above methods, the method of introducing a nucleotide sequence encoding any of the above-mentioned CD7-targeting enhanced chimeric antigen receptors into immune effector cells is selected from one or more of the following: a viral method or a non-viral method; optionally, the viral method includes using one or more of the following viral vectors: gamma retroviral vectors, lentiviral vectors, adenovirus-associated viral vectors; the non-viral method includes one or more of the following methods: gene transfer using transposons, gene transduction via mRNA, and electroporation.

[0053] In some embodiments of the above method, a nucleotide sequence encoding any of the above-described enhanced chimeric antigen receptors is introduced into immune effector cells by a method comprising the following steps:

[0054] Introducing the CRISPR / Cas system and template DNA into immune effector cells;

[0055] The CRISPR / Cas system includes the Cas protein, sgRNA and / or chRDNA targeting the CD7 gene, and sgRNA and / or chRDNA targeting the Trac gene.

[0056] The template DNA contains a nucleotide sequence encoding any of the aforementioned enhanced chimeric antigen receptors targeting CD7.

[0057] In some embodiments of the above method, the Cas protein includes one or both of Cas9 and Cas12 proteins.

[0058] In some embodiments of the above method, the sgRNA and / or chRDNA targeting the Trac gene are chRDNA sequences as shown in SEQ ID NO:25.

[0059] In some embodiments of the above method, the sgRNA and / or chRDNA targeting the CD7 gene are sgRNA sequences as shown in SEQ ID NO:26.

[0060] In some embodiments of the above method, the template DNA is dsDNA; optionally, the dsDNA sequence is as shown in SEQ ID NO:31. This application also provides the use of the above-described enhanced chimeric antigen receptor targeting CD7, isolated nucleic acid molecules, vectors, or engineered immune effector cells in the preparation of a drug for treating diseases or conditions related to CD7 expression.

[0061] In some embodiments of the above application, the disease or condition associated with CD7 expression is CD7. + Hematologic tumor.

[0062] In some embodiments of the above application, the CD7 + Hematologic malignancies are selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia (T-ALL), CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia (AML).

[0063] In some embodiments of the above application, the CD7 + Acute T-lymphoblastic leukemia includes CD7 + Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) and other acute T-cell leukemias.

[0064] In some embodiments of the above application, the CD7 + T-cell lymphoma is selected from one or more of the following: CD7 + T-lymphoblastic lymphoma (T-LBL), CD7+ extranodal NK / T-cell lymphoma, CD7 + Enteropathy-associated T-cell lymphoma, CD7 + Primary cutaneous T-cell lymphoma and CD7 + Peripheral T-cell lymphoma.

[0065] This application also provides a method for treating a disease or condition related to CD7 expression, comprising the steps of: administering an effective amount of the above-described engineered immune effector cells or pharmaceutical composition to a subject who requires treatment for a disease or condition related to CD7 expression.

[0066] In some embodiments of the above method, the disease or condition associated with CD7 expression is CD7. + Hematologic tumor.

[0067] In some embodiments of the above method, the CD7 + Hematologic malignancies are selected from one or more of the following: CD7+ Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0068] In some embodiments of the above method, the CD7 + Acute T-lymphoblastic leukemia includes CD7 + Early-stage pre-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0069] In some embodiments of the above method, the CD7 + T-cell lymphoma is selected from one or more of the following: CD7 + T-lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary cutaneous T-cell lymphoma and CD7 + Peripheral T-cell lymphoma.

[0070] In some embodiments of the above method, the administration is carried out via intravenous injection.

[0071] In some embodiments of the above method, the administration is carried out by administering an effective amount of engineered immune effector cells or a pharmaceutical composition to the subject via a single injection.

[0072] In some embodiments of the above method, the effective amount of engineered immune effector cells or drug composition is 1 × 10⁻⁶. 5 Up to 1×10 7 A dose of cells / kg.

[0073] This application also provides a medicament comprising the above-described engineered immune effector cells or pharmaceutical composition for treating diseases or conditions associated with CD7 expression.

[0074] In some embodiments of the above-mentioned drug, the disease or condition associated with CD7 expression is CD7. + Hematologic tumor.

[0075] In some embodiments of the above-mentioned drug, the CD7... + Hematologic malignancies are selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0076] In some embodiments of the above-mentioned drug, the CD7... + Acute T-lymphoblastic leukemia includes CD7 +Early-stage pre-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0077] In some embodiments of the above-mentioned drug, the CD7... + T-cell lymphoma is selected from one or more of the following: CD7 + T-lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary cutaneous T-cell lymphoma and CD7 + Peripheral T-cell lymphoma. Attached Figure Description

[0078] Figure 1 The positions of each component in the six candidate CAR structures in Example 1 are shown. SP represents the guide peptide, anti-CD7 VL and anti-CD7 VH are components of scFv, Hinge represents the hinge region, and TM represents the transmembrane region.

[0079] Figure 2 The cell viability of total T cells in each group during the culture process in Example 2 is shown.

[0080] Figure 3 The expansion fold of total T cells in each group during the culture process in Example 2 is shown.

[0081] Figure 4 This demonstrates the expansion of total T cells as effector cells in a multi-round antigen stimulation experiment using MOLT4 as the target cell in Example 3. Day 2 shows the expansion after the first round of stimulation, and Day 4 shows the single-round expansion after the second round of stimulation.

[0082] Figure 5 This demonstrates the expansion of total T cells as effector cells in a multi-round antigen stimulation experiment using CCRF as the target cells in Example 3. Day 2 shows the expansion after the first round of stimulation, and Day 4 shows the single-round expansion after the second round of stimulation.

[0083] Figure 6 This demonstrates the killing effect of effector cells on tumor cells in a multi-round antigen stimulation experiment when the target cell was MOLT4 in Example 3.

[0084] Figure 7 This demonstrates the killing effect of effector cells on tumor cells in a multi-round antigen stimulation experiment when the target cells were CCRF in Example 3.

[0085] Figure 8AThis demonstrates the expansion fold of total T cells as effector cells in the multi-round antigen stimulation experiment when the target cells were Jurkat cells in Example 3. R0 represents the initial state, and R1-R8 represent the first to the eighth rounds of antigen stimulation experiments.

[0086] Figure 8B This demonstrates the killing effect of effector cells on tumor cells in a multi-round antigen stimulation experiment when the target cells were Jurkat cells in Example 3. Round-1, round-2, round-3, round-4, round-5, round-6, round-7, and round-8 represent the first to eighth rounds of antigen stimulation experiments, respectively. Detailed Implementation

[0087] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0088] The following further describes this application: In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0089] In this application, the term "Chimeric Antigen Receptor" (CAR) is a core component of CAR cell therapy drugs, which may include an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. CAR-T (Chimeric Antigen Receptor T) cell immunotherapy is considered one of the most promising methods for conquering tumors. CAR-T cells utilize genetic modification to enable T cells to express CAR proteins. These CAR proteins are capable of recognizing intact proteins on the surface of tumor cells without relying on antigen presentation, thereby activating and functionally affecting T cells.

[0090] In this application, the term "extracellular antigen recognition domain" refers to the antigen recognition domain (ARD). CAR cell therapy products (such as CAR-T cells) rely on extracellular antigen recognition domains to specifically recognize and / or bind to target antigens expressed by tumor cells. To date, antigen recognition domains are derived from the single-chain variable fragment (scFv) of antibodies, or from receptor-ligand interactions, TCR mimics, and variable lymphocyte receptors (VLRs). To date, the most common source is scFv antibodies.

[0091] In this application, the term "specific recognition and / or binding" refers to the recognition and / or binding between the CAR extracellular antigen recognition domain and a specific target, or the recognition and / or binding between the CAR extracellular antigen recognition domain and a specific detection antibody with greater affinity, stronger affinity, easier binding, and / or longer duration than the binding of the CAR to other targets or detection antibodies.

[0092] In this application, the term "antibody" has its conventional meaning in the art and is used in the broadest sense. In the biosciences, analysis of the amino acid sequences of different antibody heavy and light chains reveals that the amino acid sequences near the N-terminus of both heavy and light chains vary considerably, while the amino acid sequences in other parts remain relatively constant. Therefore, the regions in the antibody light and heavy chains with significant amino acid sequence variation near the N-terminus are called variable regions (V), and the regions with relatively stable amino acid sequences near the C-terminus are called constant regions (C). The V regions of the heavy and light chains are abbreviated as VH and VL, respectively, and the C regions of the heavy and light chains are abbreviated as CH and CL, respectively. Within the antibody variable regions, a small subset of amino acid residues exhibits particularly strong variation; these regions, where the composition and sequence of amino acid residues are more prone to variation, are called hypervariable regions (HVRs). There are three hypervariable regions in the V regions of both the L and H chains. Because these regions can form precise complementarity with the antigenic determinants in their spatial structure, they are also called complementarity determining regions (CDRs). In antibodies, common CDR (Cellular Recognition Derivative) rules include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well-known to those skilled in the art. When using websites that apply these rules, simply inputting the VH and VL sequences and selecting the corresponding rule will yield CDR sequences based on different rules. Those skilled in the art should understand that the scope of protection of this application covers combinations of CDR sequences obtained through analysis using different rules. The six CDR regions of an antibody collectively determine its recognition ability and specificity against the corresponding antigen. Those skilled in the art should understand that when this application defines the amino acid sequences of the six CDR regions, the antibody's recognition ability and specificity against the corresponding antigen are predictable.

[0093] In this application, the term "scFv" has the conventional meaning in the art and refers to a single-chain variable region, which is an antibody composed of antibody heavy chain variable regions and light chain variable regions linked by a short peptide (linker, connecting sequence).

[0094] In this application, the term "linking sequence" generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any structure / region of the antibody or chimeric antigen receptor of the present invention. Linking sequences may consist of different amino acid residues (such as glycine and serine) to allow adjacent protein domains to move freely relative to each other. Longer linking sequences may be used when it is desirable to ensure that two adjacent domains do not interfere with each other spatially.

[0095] In this application, the term "guide peptide" refers to a recombinant protein synthesized within the cell that is exported to the extracellular space. Commonly used guide peptides include the human CD8α signal peptide or the human GM-CSF receptor α signal peptide.

[0096] In this application, the term "hinge region" refers to the connecting segment that acts between the extracellular antigen recognition domain and the transmembrane region. This region allows CAR to recognize antigens by giving the antigen recognition domain a certain range of motion.

[0097] In this application, "transmembrane region" refers to the transmembrane domain that connects the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect the expression and stability of CAR to some extent, but they do not directly participate in signal transduction; however, they can enhance downstream signal transduction through interactions.

[0098] In this application, the terms "intracellular domain," "intrinsic domain," and "intracellular region" have the same meaning, including co-stimulatory signal transduction regions, intracellular signal transduction regions, and other intracellular components directly or indirectly connected to them. In this application, the intracellular domain also includes an amino acid sequence derived from the CD7 protein. The term "intracellular signal transduction region" is responsible for activating at least one normal effector function of CAR-expressing immune effector cells. "Co-stimulatory signal transduction region" is used because, in addition to antigen-specific signal stimulation, many immune effector cells require co-stimulatory factors to promote cell proliferation, differentiation, survival, and activation of cell effector functions. In this application, the inventors explored linking other components to intracellular structures to improve the sustained expansion and tumor-killing capabilities of CAR-immune cells. In candidate enhanced CAR structures groups 1-2, the applicant added the cytokine receptor γchain (γc) to the CAR structure, and in candidate enhanced CAR structures groups 3-6, the applicant integrated the intracellular sequence of the CD7 protein into the CAR structure to verify whether these components could enhance the function of CAR-T cells.

[0099] In this application, the term "CD7 protein" refers to a transmembrane glycoprotein containing 240 amino acid residues and a member of the immunoglobulin superfamily. CD7 is widely overexpressed in immature T-cell tumors, while its expression patterns and intensity vary in mature T-cell tumors. Besides T-cell malignancies, approximately 30% of AML patients also express CD7 in leukemia blasts and malignant progenitor cells; therefore, drugs targeting CD7 can also be used to treat CD7-related diseases. + It is a mixed phenotype of AML, as well as the vast majority of NK lymphomas and NKT lymphomas. However, since T cells also express CD7, CD7 CAR-T cells can kill CD7 CAR-T "siblings" while killing tumor cells that highly express CD7.

[0100] In this application, the term "CD7 gene" has its usual meaning in the art.

[0101] In this application, the terms "Trac gene" and "TRAC gene" have their common meaning in the art, referring to the constant region of the α chain of the T cell receptor. The signal for T cell receptor (TCR) to recognize antigens is mainly transmitted through the TCR-CD3 complex (including the α chain, β chain, CD3γ, CD3δ, etc. of the TCR). The TCR is composed of α and β chains. After translation, the α and β chains assemble into a heterodimer, which binds to multiple CD3 isoform molecules. In the cell, if it cannot form a complete complex with CD3 molecules, the excess TCR will be degraded. Knocking out the Trac gene will affect the formation of the TCR-CD3 complex, meaning that the T cell receptor on the surface of T cells will be cleared, avoiding graft-versus-host disease (GVHD).

[0102] In this application, "CD7 / TRAC double-negative allogeneic T lymphocytes" refers to allogeneic T lymphocytes in which the CD7 and TRAC genes are not normally expressed, or in which the CD7 protein and the constant region of the T cell receptor α chain are not correctly expressed. Using gene editing to prevent the normal expression of CD7 on the surface of T cells is an effective method to avoid suicide injury. Simultaneously, using gene editing to prevent the normal expression of the Trac gene on T cells makes them suitable as universal CAR-T products.

[0103] In this application, the term "gene cannot be expressed normally" has the common meaning in the art. Gene expression refers to the process of synthesizing functional gene expression products from genetic information derived from genes. Gene expression products usually refer to proteins.

[0104] In this application, the terms "gene editing" and "gene editing technology" have their common meaning in the art, referring to a technique for site-specific modification of the genome. This technology allows for precise targeting to a specific site in the genome, where target DNA fragments can be cut, or target gene fragments can be knocked in or knocked out. As a molecular biology technique, gene editing technology enables precise modification of chromosomes, thereby altering the existing functions of cells. Compared to cell lines commonly used in basic research, T cells, as primary cells, are not particularly unique except for their inability to proliferate long-term, and can also be edited using gene editing technology. Currently, there are three main gene editing tools: zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, and RNA-guided CRISPR / Cas nuclease technology. Compared to traditional gene targeting techniques, new gene editing technologies retain the characteristic of site-specific modification, can be applied to more species and cells, are more efficient, have shorter construction times, and are less costly.

[0105] In this application, the terms "CRISPR / Cas technology" and "CRISPR / Cas system" have their common meaning in the art. CRISPR stands for clustered regularly interspaced shortpalindromic repeats / CRISPR-associated proteins, an acquired immune system found in most bacteria and all archaea that can directionally cleave foreign gene fragments. Different types of CRISPR / Cas systems have been discovered, with the second type being relatively simple, consisting primarily of Cas proteins and guide RNA (gRNA, also known as sgRNA). Compared to earlier ZFN and TALEN technologies, CRISPR / Cas offers advantages such as low off-target rates, high efficiency, affordability, and wide applicability. Furthermore, some literature reports that CRISPR hybrid RNA-DNA (chRDNA) guidance technology significantly improves the specificity of the Cas9 protein compared to whole RNA guidance technology, thereby achieving high levels of intended genome editing in cells and minimizing off-target effects.

[0106] In this application, the terms "sequentially linked," "sequentially connected," and "-" have similar meanings, all indicating the connection of different parts in a specific order, without limiting the connection to direct or indirect linking. In the description of amino acid sequences, unless specifically indicated, the direction of connection is from the N-terminus to the C-terminus, as is commonly used in the industry; in the description of nucleotide sequences, unless specifically indicated, the direction of connection is from the 5' end...

[0107] The direction from end 3' to end 4'. Similarly, the expressions "connected" and "linked" do not specify whether they are directly connected or indirectly connected.

[0108] In this application, the term "separated" generally refers to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" substance, it may be due to a change in its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated substance. The term "separated" does not exclude substances obtained artificially from their natural state and then synthesized, nor does it exclude the presence of other impurities that do not affect the substance's activity.

[0109] In this application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotide, deoxyribonucleotide, or ribonucleotide of any length, which may be isolated from its natural environment or an analogue synthesized artificially.

[0110] The term "vector" generally refers to a nucleic acid delivery vehicle that inserts a polynucleotide encoding a protein into itself, thereby enabling the protein to be expressed. Vectors can transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed within the host cell. Examples of vectors include: plasmids; phage particles; Cos plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain replication initiation sites. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances. The term "transposon" refers to a discontinuous segment of DNA capable of migrating between chromosomal loci and carrying genetic information, such as the Sleeping Beauty SB system and the PB system derived from lepidopteran insects. In some embodiments, electroporation can also be used to transfect mRNA into T cells.

[0111] In this application, the terms "comprising" or "including" generally mean including the explicitly specified features, but do not exclude other elements.

[0112] In this application, the term "about" generally refers to a range of fluctuation acceptable to a person skilled in the art above or below a specified value, such as a variation within ±0.5% to 10%, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, or more above or below the specified value.

[0113] Variations within the range of 8.5%, 9%, 9.5%, or 10%.

[0114] Enhanced chimeric antigen receptor

[0115] This application provides an enhanced chimeric antigen receptor comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. The intracellular domain comprises a co-stimulatory signal transduction region and an intracellular signal transduction region, wherein the intracellular domain further comprises an amino acid sequence derived from the CD7 protein.

[0116] In some embodiments of the aforementioned enhanced chimeric antigen receptor, the amino acid sequence derived from the CD7 protein is an amino acid sequence derived from the intracellular region of the CD7 protein.

[0117] In some embodiments of the enhanced chimeric antigen receptor described above, the amino acid sequence derived from the intracellular region of the CD7 protein comprises the amino acid sequence shown in SEQ ID NO:16; optionally, the amino acid sequence derived from the intracellular region of the CD7 protein is shown in SEQ ID NO:16.

[0118] In some embodiments of the aforementioned enhanced chimeric antigen receptor, an amino acid sequence derived from the CD7 protein is linked between the transmembrane region and the co-stimulatory signal transduction region.

[0119] In some embodiments of the enhanced chimeric antigen receptor described above, the co-stimulatory signal transduction region comprises an amino acid sequence derived from 4-1BB or CD28; optionally, the co-stimulatory signal transduction region comprises an amino acid sequence derived from 4-1BB; more preferably, the co-stimulatory signal transduction region is an amino acid sequence derived from 4-1BB.

[0120] And / or, wherein the intracellular signal transduction region comprises one or more amino acids derived from CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signal transduction region comprises an amino acid sequence derived from CD3ζ; further optionally, the intracellular signal transduction region is an amino acid sequence derived from CD3ζ.

[0121] In some embodiments of the aforementioned enhanced chimeric antigen receptor, the amino acid sequence derived from 4-1BB includes the amino acid sequence shown in SEQ ID NO:13; optionally, the amino acid sequence derived from 4-1BB is as follows:

[0122] As shown in SEQ ID NO:13.

[0123] In some embodiments of the aforementioned enhanced chimeric antigen receptor, the amino acid sequence derived from CD3ζ includes the amino acid sequence shown in SEQ ID NO:15; optionally, the amino acid sequence derived from CD3ζ is as follows:

[0124] As shown in SEQ ID NO:15.

[0125] In some embodiments of the enhanced chimeric antigen receptor described above, the intracellular domain includes, as shown in SEQ ID NO:

[0126] The amino acid sequence shown in NO:21; optionally, the amino acid sequence of the intracellular domain is as shown in SEQ ID.

[0127] Shown in NO:21.

[0128] In some embodiments of the aforementioned enhanced chimeric antigen receptor, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, CD8α, and DAP10; optionally, the amino acids in the hinge region are derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises, as shown in SEQ ID NO.

[0129] The amino acid sequence shown in NO:11; more preferably, the amino acid sequence of the hinge region is as shown in SEQ ID.

[0130] Shown in NO:11.

[0131] In some embodiments of the enhanced chimeric antigen receptor described above, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70, DAP10, 2B4, DNAM-1, and HVEM; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:12; even more optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:12.

[0132] In some embodiments of the enhanced chimeric antigen receptor described above, a guide peptide is further attached to the N-terminus of the extracellular antigen recognition domain; optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO:1; further optionally, the amino acid sequence of the guide peptide is shown in SEQ ID NO:1.

[0133] Isolated nucleic acid molecules, vectors, immune effector cells, and drug compositions

[0134] This application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the above-mentioned enhanced chimeric antigen receptors.

[0135] This application also provides a vector containing the isolated nucleic acid molecules described above.

[0136] This application also provides an engineered immune effector cell comprising the above-described enhanced chimeric antigen receptor, the above-described isolated nucleic acid molecule, or the above-described carrier.

[0137] In some embodiments, the engineered immune effector cells described above are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0138] In some embodiments, the engineered immune effector cells described above are T lymphocytes; alternatively, the T lymphocytes are autologous T lymphocytes or allogeneic T lymphocytes.

[0139] In some embodiments, the engineered immune effector cells described above are αβT lymphocytes or γδT lymphocytes.

[0140] This application also provides a pharmaceutical composition comprising the above-described engineered immune effector cells and pharmaceutically acceptable excipients.

[0141] In some embodiments of the above pharmaceutical compositions, pharmaceutically acceptable excipients include protective agents.

[0142] In some embodiments of the above pharmaceutical compositions, pharmaceutically acceptable excipients include cell cryopreservation solutions.

[0143] In some embodiments, the pharmaceutical composition described above is an intravenous injection.

[0144] Methods for preparing engineered immune effector cells

[0145] This application also provides a method for preparing engineered immune effector cells, comprising the following steps: introducing a nucleotide sequence encoding any of the above-mentioned enhanced chimeric antigen receptors into the immune effector cells.

[0146] In some embodiments of the above method, the immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0147] In some embodiments of the above method, the immune effector cells are T lymphocytes; optionally, the T lymphocytes are autologous T lymphocytes or allogeneic T lymphocytes.

[0148] In some embodiments of the above method, the T lymphocytes are αβT lymphocytes or γδT lymphocytes.

[0149] In some embodiments of the above methods, the method of introducing a nucleotide sequence encoding any of the chimeric antigen receptors into immune effector cells is selected from one or more of the following: a viral method or a non-viral method; optionally, the viral method includes using one or more of the following viral vectors: gamma retroviral vector, lentiviral vector, adenovirus-associated viral vector; the non-viral method includes one or more of the following methods: gene transfer using transposons, gene transduction via mRNA, and electroporation.

[0150] In some embodiments of the above method, a nucleotide sequence encoding any of the above-described enhanced chimeric antigen receptors is introduced into immune effector cells by a method comprising the following steps:

[0151] Introducing the CRISPR / Cas system and template DNA into immune effector cells;

[0152] The CRISPR / Cas system includes: a Cas protein, and sgRNA and / or chRDNA that target the insertion site of the nucleotide sequence encoding any of the above-mentioned enhanced chimeric antigen receptors;

[0153] The template DNA contains a nucleotide sequence encoding any of the aforementioned enhanced chimeric antigen receptors.

[0154] In some embodiments of the above method, the Cas protein includes one or both of Cas9 and Cas12 proteins.

[0155] Applications and drugs

[0156] This application also provides the use of the above-mentioned enhanced chimeric antigen receptor, isolated nucleic acid molecule, carrier, or engineered immune effector cell in the preparation of a drug.

[0157] This application also provides a medicament comprising the above-described engineered immune effector cells or pharmaceutical composition for treating diseases or conditions.

[0158] Enhanced chimeric antigen receptor targeting CD7

[0159] This application provides an enhanced chimeric antigen receptor targeting CD7, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain targeting CD7. The intracellular domain includes a co-stimulatory signal transduction region and an intracellular signal transduction region, wherein the intracellular domain further comprises an amino acid sequence derived from the CD7 protein.

[0160] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the amino acid sequence derived from the CD7 protein is an amino acid sequence derived from the intracellular region of the CD7 protein.

[0161] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the amino acid sequence derived from the intracellular region of the CD7 protein comprises the amino acid sequence shown in SEQ ID NO:16; optionally, the amino acid sequence derived from...

[0162] The amino acid sequence of the intracellular region of the CD7 protein is shown in SEQ ID NO:16.

[0163] In some embodiments, the aforementioned enhanced chimeric antigen receptor targeting CD7 has an amino acid sequence derived from the CD7 protein linked between the transmembrane region and the co-stimulatory signal transduction region.

[0164] In some embodiments of the aforementioned CD7-targeting enhanced chimeric antigen receptor, the co-stimulatory signal transduction region comprises an amino acid sequence derived from 4-1BB or CD28; optionally, the co-stimulatory signal transduction region comprises an amino acid sequence derived from 4-1BB; further optionally, the co-stimulatory signal transduction region is an amino acid sequence derived from 4-1BB.

[0165] And / or, wherein the intracellular signal transduction region comprises one or more amino acids derived from CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signal transduction region comprises an amino acid sequence derived from CD3ζ; further optionally, the intracellular signal transduction region is an amino acid sequence derived from CD3ζ.

[0166] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the amino acid sequence derived from 4-1BB includes the amino acid sequence shown in SEQ ID NO:13; optionally, the amino acid sequence derived from 4-1BB is shown in SEQ ID NO:13.

[0167] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the amino acid sequence derived from CD3ζ includes the amino acid sequence shown in SEQ ID NO:15; optionally, the amino acid sequence derived from CD3ζ is shown in SEQ ID NO:15.

[0168] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the intracellular domain comprises the amino acid sequence shown in SEQ ID NO:21; alternatively, the amino acid sequence of the intracellular domain is as follows:

[0169] As shown in SEQ ID NO:21.

[0170] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the extracellular antigen recognition domain targeting CD7 comprises a CD7 antibody heavy chain variable region and a CD7 antibody light chain variable region. The amino acid sequences of CDR1, CDR2, and CDR3 in the CD7 antibody heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CD7 antibody light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody light chain variable region shown in SEQ ID NO:4. Optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the CD7 antibody heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 in the CD7 antibody light chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in SEQ ID NO:4. The amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the antibody light chain shown in NO:4.

[0171] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CD7 antibody heavy chain respectively comprise the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CD7 antibody light chain respectively comprise the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CD7 antibody heavy chain are as shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the CD7 antibody light chain are as shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.

[0172] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the variable region of the CD7 antibody heavy chain contains the amino acid sequence shown in SEQ ID NO:3, and the variable region of the CD7 antibody light chain contains the amino acid sequence shown in SEQ ID NO:4; optionally, the amino acid sequence of the variable region of the CD7 antibody heavy chain is as shown in SEQ ID NO:3, and the amino acid sequence of the variable region of the CD7 antibody light chain is as shown in SEQ ID NO:4.

[0173] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the extracellular antigen recognition domain targeting CD7 comprises a humanized CD7 scFv antibody.

[0174] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the extracellular antigen recognition domain targeting CD7 comprises the amino acid sequence shown in SEQ ID NO:2; optionally, the extracellular antigen recognition domain targeting CD7 is the amino acid sequence shown in SEQ ID NO:2.

[0175] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, CD8α, and DAP10; optionally, the amino acids in the hinge region are derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:11; even more optionally, the amino acid sequence of the hinge region is as shown in SEQ ID NO:11.

[0176] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70, DAP10, 2B4, DNAM-1, and HVEM; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:12; even more optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:12.

[0177] In some embodiments of the aforementioned enhanced chimeric antigen receptor targeting CD7, a guide peptide is further attached to the N-terminus of the extracellular antigen recognition domain; optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO:1; further optionally, the amino acid sequence of the guide peptide is shown in SEQ ID NO:1.

[0178] In some embodiments, the enhanced chimeric antigen receptor targeting CD7 described above has an amino acid sequence as shown in SEQ ID NO:1-SEQ ID NO:2-SEQ ID NO:11-SEQ ID NO:12-SEQ ID NO:21, with the amino acids linked in order from the N-terminus to the C-terminus; optionally, the sequential linking is a direct linking.

[0179] Isolated nucleic acid molecules, vectors, immune effector cells, and drug compositions

[0180] This application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the aforementioned enhanced chimeric antigen receptors targeting CD7.

[0181] This application also provides a vector containing the isolated nucleic acid molecules described above.

[0182] This application also provides an engineered immune effector cell comprising the above-described enhanced chimeric antigen receptor targeting CD7, the above-described isolated nucleic acid molecule, or the above-described carrier.

[0183] In some embodiments, the engineered immune effector cells described above are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0184] In some embodiments, the engineered immune effector cells described above are T lymphocytes; alternatively, the T lymphocytes are autologous T lymphocytes or allogeneic T lymphocytes.

[0185] In some embodiments, the allogeneic T lymphocytes described above comprise CD7 / TRAC double-negative cells obtained through gene editing; further, the allogeneic T lymphocytes comprise 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, or 99% or more of CD7 / TRAC double-negative cells obtained through gene editing.

[0186] In some embodiments, the engineered immune effector cells described above are αβT lymphocytes or γδT lymphocytes.

[0187] This application also provides a pharmaceutical composition comprising the above-described engineered immune effector cells and pharmaceutically acceptable excipients.

[0188] In some embodiments of the above pharmaceutical compositions, pharmaceutically acceptable excipients include protective agents.

[0189] In some embodiments of the above pharmaceutical compositions, pharmaceutically acceptable excipients include cell cryopreservation solutions.

[0190] In some embodiments, the pharmaceutical composition described above is an intravenous injection.

[0191] Methods for preparing engineered immune effector cells targeting CD7

[0192] This application also provides a method for preparing engineered immune effector cells that target CD7, comprising the following steps: introducing a nucleotide sequence encoding any of the above-mentioned enhanced chimeric antigen receptors that target CD7 into the immune effector cells.

[0193] In some embodiments of the above method, the immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0194] In some embodiments of the above method, the immune effector cells are T lymphocytes; optionally, the T lymphocytes are autologous T lymphocytes or allogeneic T lymphocytes.

[0195] In some embodiments of the above method, the T lymphocytes are αβT lymphocytes or γδT lymphocytes.

[0196] In some embodiments of the above methods, the method of introducing a nucleotide sequence encoding any of the chimeric antigen receptors into immune effector cells is selected from one or more of the following: a viral method or a non-viral method; optionally, the viral method includes using one or more of the following viral vectors: gamma retroviral vector, lentiviral vector, adenovirus-associated viral vector; the non-viral method includes one or more of the following methods: gene transfer using transposons, gene transduction via mRNA, and electroporation.

[0197] In some embodiments of the above method, a nucleotide sequence encoding any of the above-described enhanced chimeric antigen receptors is introduced into immune effector cells by a method comprising the following steps:

[0198] Introducing the CRISPR / Cas system and template DNA into immune effector cells;

[0199] The CRISPR / Cas system includes the Cas protein, sgRNA and / or chRDNA targeting the CD7 gene, and sgRNA and / or chRDNA targeting the Trac gene.

[0200] The template DNA contains a nucleotide sequence encoding any of the aforementioned enhanced chimeric antigen receptors targeting CD7.

[0201] In some embodiments of the above method, the Cas protein includes one or both of Cas9 and Cas12 proteins.

[0202] In some embodiments of the above method, the sgRNA and / or chRDNA targeting the Trac gene are chRDNA sequences as shown in SEQ ID NO:25.

[0203] In some embodiments of the above method, the sgRNA and / or chRDNA targeting the CD7 gene are sgRNA sequences as shown in SEQ ID NO:26.

[0204] In some embodiments of the above method, the template DNA is dsDNA; optionally, the dsDNA sequence is as follows:

[0205] As shown in SEQ ID NO:31.

[0206] Applications, treatment methods and drugs

[0207] This application also provides the use of the above-mentioned enhanced chimeric antigen receptor targeting CD7, isolated nucleic acid molecules, vectors, or engineered immune effector cells in the preparation of a drug for treating diseases or conditions related to CD7 expression.

[0208] In some embodiments of the above application, the disease or condition associated with CD7 expression is CD7. + Hematologic tumor.

[0209] In some embodiments of the above application, the CD7 + Hematologic malignancies are selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0210] In some embodiments of the above application, the CD7 + Acute T-lymphoblastic leukemia includes CD7 + Early-stage pre-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0211] In some embodiments of the above application, the CD7+ T-cell lymphoma is selected from one or more of the following: CD7 + T-lymphoblastic lymphoma, CD7+ extranodal NK / T-cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary cutaneous T-cell lymphoma and CD7 + Peripheral T-cell lymphoma.

[0212] This application also provides a method for treating a disease or condition related to CD7 expression, comprising the steps of: administering an effective amount of the above-described engineered immune effector cells or pharmaceutical composition to a subject who requires treatment for a disease or condition related to CD7 expression.

[0213] In some embodiments of the above method, the disease or condition associated with CD7 expression is CD7. + Hematologic tumor.

[0214] In some embodiments of the above method, the CD7 + Hematologic malignancies are selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0215] In some embodiments of the above method, the CD7 + Acute T-lymphoblastic leukemia includes CD7 + Early-stage pre-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0216] In some embodiments of the above method, the CD7 + T-cell lymphoma is selected from one or more of the following: CD7 + T-lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary cutaneous T-cell lymphoma and CD7 + Peripheral T-cell lymphoma.

[0217] In some embodiments of the above method, the administration is performed via intravenous injection.

[0218] In some embodiments of the above method, the administration is carried out by administering an effective amount of engineered immune effector cells or a pharmaceutical composition to the subject via a single injection.

[0219] In some embodiments of the above method, the effective amount of engineered immune effector cells or drug composition is 1 × 10⁻⁶. 5Up to 1×10 7 A dose of cells / kg.

[0220] This application also provides a medicament comprising the above-described engineered immune effector cells or pharmaceutical composition for treating diseases or conditions associated with CD7 expression.

[0221] In some embodiments of the above-mentioned drugs, the disease or condition associated with CD7 expression is CD7. + Hematologic tumor.

[0222] In some embodiments of the above-mentioned drug, the CD7... + Hematologic malignancies are selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

[0223] In some embodiments of the above-mentioned drug, the CD7... + Acute T-lymphoblastic leukemia includes CD7 + Early-stage pre-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

[0224] In some embodiments of the above-mentioned drug, the CD7... + T-cell lymphoma is selected from one or more of the following: CD7 + T-lymphoblastic lymphoma, CD7+ extranodal NK / T cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary cutaneous T-cell lymphoma and CD7 + Peripheral T-cell lymphoma.

[0225] Not intended to be limited by any theory, the embodiments described below are merely for illustrating the chimeric antigen receptor, immune effector cells, preparation methods, and uses of this application, and are not intended to limit the scope of the invention. The embodiments do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) *Molecular Cloning: A Laboratory Manual*, 2nd edition, Cold Spring Harbor Laboratory Press.

[0226] Example 1: Preparation of CAR-T cells with enhanced CAR structure

[0227] 1. Design of Enhanced CAR Structure

[0228] The inventors constructed six enhanced CAR structures as candidates and two second-generation CAR structures as positive controls, selecting the most effective enhanced CAR structure through functional validation on T cells. The positions of the components in each candidate CAR structure are shown below. Figure 1 As shown: the amino acid sequence of the guide peptide is shown in SEQ ID NO:1; the scFv targeting CD7 serves as the extracellular antigen recognition domain, and its amino acid sequence is shown in SEQ ID NO:2 (wherein: in the scFv targeting CD7, the amino acid sequence of CD7 VH is shown in SEQ ID NO:3, and the amino acid sequence of CD7 VL is shown in SEQ ID NO:4); the amino acid sequence of the hinge region derived from CD8α is shown in SEQ ID NO:11; the amino acid sequence of the transmembrane region derived from CD8α is shown in SEQ ID NO:12; the amino acid sources and specific sequences of the intracellular domains in each candidate enhanced CAR structure and positive control CAR structure are shown in Table 1.

[0229] Table 1. Amino acid sequences of intracellular domains in each CAR structure

[0230] CAR structural grouping Intracellular domain amino acid sources amino acid sequence Positive control CAR structure group 1 4-1BB-CD3ζ SEQ ID NO:17 Positive control CAR structure 2 groups CD28-CD3ζ SEQ ID NO:18 One group of candidate enhanced CAR structures 4-1BB-CD3ζ-γc SEQ ID NO:19 Two candidate enhanced CAR structures CD28-CD3ζ-γc SEQ ID NO:20 Three candidate enhanced CAR structures CD7-4-1BB-CD3ζ SEQ ID NO:21 Four candidate enhanced CAR structures 4-1BB-CD7-CD3ζ SEQ ID NO:22 Five candidate enhanced CAR structures CD7-CD28-CD3ζ SEQ ID NO:23 Six candidate enhanced CAR structures CD28-CD7-CD3ζ SEQ ID NO:24

[0231] Table 1 shows the origins of each part of the intracellular domain, focusing only on the origin and relative position of each part of the intracellular domain sequence, as shown in the amino acid sequence.

[0232] In candidate enhanced CAR structures 1-2, the applicant incorporated the cytokine receptor γchain (γc) into the CAR structure to verify whether γc could enhance the function of CAR-T cells. γc is a common subunit of IL2 and IL15, and studies have shown that it can increase the secretion of IL2, which is a key cytokine for promoting T cell proliferation and performing cytotoxic functions.

[0233] In candidate enhanced CAR structures 3-6, the applicant integrated the intracellular region sequence of the CD7 protein into the CAR structure, so that the CAR expressed by T cells contains the CD7 intracellular protein region, in order to verify whether the CD7 intracellular protein region can enhance the function of CAR-T cells. CD7 in T cells can promote the production of IL2, and IL2 is a key cytokine for promoting T cell proliferation and performing cytotoxic functions.

[0234] 2. The preparation method of CAR-T cells is as follows:

[0235] 1) Sorting T cells

[0236] Peripheral blood mononuclear cells (PBMCs) are isolated from human apheresis cells, and then T cells are sorted from the PBMCs.

[0237] 2) T cells were activated by magnetic beads.

[0238] The isolated T cells were resuspended in complete lymphocyte culture medium (OpTmizer CTS + 2.6% OpTmizer supplement + 5% ISR + 1% GlutaMax-1 + 10 ng / ml IL-7 + 5 ng / ml IL-15) to a final concentration of 1E6–3E6 cells / ml, and then administered at a concentration of 1 μl beads / 1×10⁻⁶. 6 T cells were stimulated and activated by adding CD3 / CD28 magnetic beads (purchased through commercial channels), and then incubated in an incubator at 37°C + 5% CO2 for at least 24 hours to obtain activated T cells.

[0239] 3) Gene editing of activated T cells.

[0240] The CD7 and TRAC genes are rendered unexpressed using the CRISPR / Cas9 system. Simultaneously, a nucleotide sequence encoding a CAR is knocked into the CD7 gene site using non-viral site-directed integration technology. The principle behind this process is as follows: Guided by sgRNA, the Cas9 endonuclease cleaves the DNA double strand at a specific site, creating a DNA gap. A template DNA segment (Donor DNA) is then introduced into T cells via a non-viral vector. The Donor DNA contains left and right homologous arms highly homologous to sequences near the DNA gap, as well as nucleotides encoding the CAR sequence. Homologous recombination repair of the DNA gap uses the Donor DNA as a template, thus achieving the goal of site-directed introduction of the target fragment into the genome. The specific steps are as follows:

[0241] After removing the magnetic beads from the activated T cells, centrifuging to completely remove the supernatant, and then using electroporation buffer (derived from Nucleofector), the cells were subjected to electroporation. TM The kit contains Nucleofector TM The cell suspension was prepared by resuspending cells in 81.8 μL of Nucleofector solution and 18.2 μL of additives.

[0242] Mix Cas9 protein, chRDNA targeting the Trac gene, and sgRNA targeting the CD7 gene, and incubate in electroporation buffer at room temperature for 15 minutes. After incubation, construct the gene editing system according to the proportions in Table 2: Add dsDNA as template DNA and cell suspension to the gene editing system, mix well, and electroporate. Then, quickly add an appropriate amount of T cell complete medium-E (90% T cell complete medium + 10% FBS + 1 μM M3814, where T cell complete medium refers to X-VIVO15 medium supplemented with 5 ng / ml IL-15 + 10 ng / ml IL-7, and M3814 refers to Nedisertib, a highly selective DNA-activating protein kinase inhibitor) to the gene editing system, and incubate at 37℃ + 5% CO2 for 15-30 minutes.

[0243] Table 2. Gene Editing System

[0244] Gene editing system Preparation of concentration Dosage sequence Cell count --- 1×10E6 --- Cas9 10mg / mL 0.3μL --- chRDNA targeting the Trac gene 0.1 nmol / μl 0.45μL SEQ ID NO:25 sgRNA targeting the CD7 gene 0.1 nmol / μl 0.45μL SEQ ID NO:26 Template dsDNA --- 0.5μg ---

[0245] In Table 2, the template dsDNA sequences in positive control CAR structure group 1, positive control CAR structure group 2, candidate enhanced CAR structure group 1, candidate enhanced CAR structure group 2, candidate enhanced CAR structure group 3, candidate enhanced CAR structure group 4, candidate enhanced CAR structure group 5, and candidate enhanced CAR structure group 6 are shown as SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34, respectively.

[0246] Two negative control groups were set up: T cells that knocked out both the Trac and CD7 genes but did not integrate CAR at a specific site (dKO-UTD group) and T cells that did not knock out both the Trac and CD7 genes and did not integrate CAR at a specific site (Noep group). The dKO-UTD group was also obtained using the above method, but template dsDNA was not added to its gene editing system.

[0247] 4) Expansion culture of cells in each group

[0248] After incubation, T cell complete culture medium-E was added to adjust the cell density to 5×10E6 / mL, and the cells were placed in an incubator to continue culturing at 37℃ + 5% CO2. After 24 hours, T cell complete culture medium-E was added to adjust the cell density to 2×10E6 / mL for use in subsequent examples.

[0249] The obtained CAR-T cells were named according to the amino acid source of their intracellular domains. For example, CAR-T cells with an intracellular domain amino acid source of CD7-4-1BB-CD3ζ were named CD7-4-1BB-CD3ζ CAR-T cells, and those with an intracellular domain amino acid source of CD7-CD28-CD3ζ were named CD7-CD28-CD3ζ CAR-T cells. The six enhanced CAR-T cell types were compared with two second-generation CAR-T cell types used as positive controls. The viability, expansion capacity, sustained expansion after multiple rounds of antigen stimulation, and sustained killing ability of the CAR-T cells were evaluated to screen for the optimal enhanced CAR structure.

[0250] Example 2: Viability and Expansion Capacity of CAR-T Cells

[0251] Samples were taken from six candidate enhanced CAR-T cells, two positive control CAR-T cells, double-knockout but non-targeted CAR-integration T cells (dKO-UTD group), and non-double-knockout and non-targeted CAR-integration T cells (Noep group) on days 3, 5, 8, 10, and 12 after electroporation using AO / PI dual fluorescence staining (AO / PI dual staining apoptosis detection kits are available commercially and use AO / PI probes to double-stain cell nuclei to detect the state of apoptotic cells, which can distinguish between normal cells and apoptotic cells). Cells in each group were counted using a cell counter to calculate the total T cell viability and expansion fold during the culture process.

[0252] The viability of total T cells at different days after electroporation under the cell amplification and culture conditions in Example 1 is as follows: Figure 2 As shown: There was no significant difference in cell viability among different groups at different days after electroporation. Specifically, on day 12 after electroporation, the cell viability of all six enhanced CAR-T cell types, two positive control CAR-T cell types, and double-knockout T cells that did not integrate CAR at specific sites remained above 90%. Under the cell expansion culture conditions in Example 1, the fold increase in total T cells at different days after electroporation is as follows: Figure 3 As shown, on day 12 after electroporation, the total T cell expansion fold of the four CD7-enhanced groups and the CD28-CD3ζ-γc group was about twice that of the two positive control groups.

[0253] Example 3: Multiple rounds of antigen stimulation (Stress-Test) experiment

[0254] Antigen stimulation can activate CAR-T cells and promote their proliferation. However, continuous activation of T cells can lead to cell exhaustion. Exhausted T cells will have reduced proliferative capacity and effector function. By detecting the proliferation and sustained killing capacity of total T cells after multiple rounds of antigen stimulation experiments, the ability to proliferate and kill for a long time is used as the preferred criterion.

[0255] To evaluate the sustained expansion and tumor-killing ability of CAR-T cells, cells cultured for 8 days after electroporation were used for multiple rounds of stimulation experiments. The experimental procedure was as follows: Molt4 cells (human acute lymphoblastic leukemia cells) expressing GFP fluorescent protein and CCRF-CEM cells (human acute lymphoblastic leukemia cells) were selected as target cells. The first round of antigen stimulation was performed in X-VIVO + 5% FBS + 1% GlutaMAX medium at a ratio of 1:2 for effector cells (CAR+ cells). Forty-eight hours later, the second round of antigen stimulation was performed, using cells from the first round at a ratio of 2:1 for effector cells (total T cells). After each round, flow cytometry was used to detect the number of T cells without GFP fluorescence and the number of target cells with GFP fluorescence. The fold increase in total T cells and the proportion of residual tumor cells in that round were calculated.

[0256] Total T cell expansion in multiple rounds of antigen stimulation experiments, as follows Figure 4 and Figure 5 As shown: In the two rounds of multi-round stimulation experiments, among the six enhanced CAR-T cell groups, the CD7-4-1BB-CD3ζ group showed the highest fold increase in total T cells, and the CD7-4-1BB-CD3ζ group exhibited superior sustained expansion capacity. Specifically: when the target cell was MOLT4, the CD7-4-1BB-CD3ζ group showed a 5-fold increase in total T cells per round over two cycles compared to the non-enhanced positive control group; when the target cell was CCRF, the CD7-4-1BB-CD3ζ group showed a 2.37-fold increase in total T cells per round over two cycles compared to the non-enhanced positive control group. The tumor cell killing effect in the multi-round antigen stimulation experiments is as follows: Figure 6 and Figure 7As shown: When the target cell was MOLT4, the 4-1BB-CD3ζ group (with a residual tumor cell rate close to 0% as seen in the bar chart), the 4-1BB-CD3ζ-γc group (with a residual tumor cell rate close to 0% as seen in the bar chart), and the CD7-4-1BB-CD3ζ group had the lowest mean residual target cell rates, indicating that these three groups had stronger sustained killing ability against MOLT4. When the target cell was CCRF, the 4-1BB-CD3ζ group and the CD7-4-1BB-CD3ζ group had the lowest mean residual target cell rates, indicating that they had stronger sustained killing ability against CCRF. Considering both sustained amplification and sustained tumor killing ability in the above multi-round antigen stimulation experiments, the CD7-4-1BB-CD3ζ group was the best.

[0257] To further validate the advantages of the CD7-4-1BB-CD3ζ group in multi-round antigen stimulation experiments, more rounds of antigen stimulation experiments were conducted using a third type of target cells. Jurkat cells (acute T-lymphoblastic leukemia cells) expressing GFP fluorescent protein were selected as target cells. Antigen stimulation was performed in X-VIVO + 5% FBS + 1% GlutaMAX medium at a 1:1 ratio of effector cells (counted as CAR+ cells). Every two days, 500 μL of sample was collected for the next round of plate formation, and target cells were added to maintain a 1:1 ratio. After each round, flow cytometry was used to detect the number of T cells without GFP fluorescence and the number of target cells with GFP fluorescence. The total T cell expansion fold and the proportion of residual tumor cells were calculated for that round. The total T cell expansion capacity and tumor cell killing effect are shown below. Figure 8A and Figure 8B As shown: After 8 rounds of stimulation, the total T cell expansion fold of the CD7-4-1BB-CD3ζ group was 6.26 times that of the non-enhanced positive control group, indicating that its sustained expansion capacity was stronger; in addition, during the 8 rounds of stimulation, the target cells in both the CD7-4-1BB-CD3ζ group and the non-enhanced positive control group were completely killed, and there was no significant difference between the two.

[0258] Example 4: In vivo experiments in mice

[0259] Experimental Methods: Jurkat-GFP-Luc cells (Jurkat cells were infected with lentiviruses containing nucleotide sequences encoding GFP protein and luciferase, and the resulting cells were then identified to obtain cell lines stably expressing GFP protein and luciferase. Luciferase is used to provide a fluorescence signal; for the same cell line, the weaker the fluorescence signal, the fewer tumor cells remain and the stronger the killing effect. The luciferase gene is located in GenBank: MK484106.1; the GFP protein sequence is known and is used for convenient selection of stable cell lines) were used at a rate of 1 × 10⁻⁶. 6Tumor-bearing mice were injected with 0.2 mL of the drug via the tail vein. On day 4 post-inoculation, when the average tumor imaging signal intensity was 3.50E+06p / sec, the mice were randomly assigned to four groups based on tumor imaging signal intensity and body weight: PBS group, dKO UTD group, second-generation CAR structure group, and enhanced CAR structure group. The PBS and dKO UTD groups each contained three mice, while the second-generation CAR structure and enhanced CAR structure groups each contained four mice.

[0260] Table 3. Grouping of mice in vivo experiments

[0261]

[0262] All groups were administered via a single tail vein injection. Mice were continuously monitored after reinfusion, with mouse death serving as the endpoint. The observation period lasted until day 118. During the observation period following administration (days 0, 3, 7, 10, 14, 21, 28, 35, 42, and 112), in vivo imaging and mouse weight were performed twice weekly. Mouse weight and tumor imaging signal intensity were recorded, and the differences in tumor suppression effects among groups in the mouse model were assessed based on the imaging signal intensity. At the end of the experiment, the survival time of each group was recorded.

[0263] Experimental Results: The body weight of animals in all groups decreased slightly in the early stages of the experiment, then increased subsequently. As the tumor burden increased, body weight decreased again. The animals tolerated the drug well. At day 0, the mean tumor signal value in group G1 was 4.24E+06p / sec, and the mean tumor signal values ​​in groups G2-G4 were 4.11E+06p / sec, 4.16E+06p / sec, and 4.26E+06p / sec, respectively. After drug administration, tumor inhibition was observed in groups G3-G4 starting on day 3. The mean tumor signal value in group G1 was 7.36E+06p / sec, and the mean tumor signal values ​​in groups G2-G4 were 7.51E+06p / sec, 2.44E+06p / sec, and 1.77E+06p / sec, respectively. Compared with the G1 PBS group, the tumor inhibition rates of the dKO UTD group, the second-generation CAR structure group, and the enhanced CAR structure group were -8.9%, 155.2%, and 179.6%, respectively. Tumor inhibition rate = 1 - (mean signal value of the experimental group) T -Average signal value of the experimental group T0 ) / (average signal value of control group 1) T - Average signal value of control group 1 T0Except for the G2 dKO UTD group, which served as the negative control group, both the G3 second-generation CAR structure group and the G4 enhanced CAR structure group showed significant tumor suppression effects. With continued observation after infusion, differences in tumor suppression began to emerge among the groups. In this experiment, the median survival of animals in the G1 PBS group was 45 days, while the median survival of animals in the G2-G4 groups was 45, 63, and 99 days, respectively (Log-rank (Mantel-Cox) test P value < 0.0001). Both the G3 second-generation CAR structure group and the G4 enhanced CAR structure group significantly prolonged the survival of tumor-bearing animals, with the G4 enhanced CAR structure group showing better performance.

[0264] Sequence Description

[0265] SEQ ID NO:1---Guide peptide amino acid sequence;

[0266] MALPVTALLLPLALLLHAARP

[0267] The amino acid sequence of SEQ ID NO:2---CD7 scFv;

[0268] DIQMTQSPSSSLSASVGDRVTITCSASQGISNYLNWYQQKPDKTVKLLIYYTSSLHSG

[0269] VPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKLEIKGGGGSGG

[0270] GGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGLTFSSYAMSWVRQAPEKRLEW

[0271] VSSISSGGFTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDEVRGY

[0272] LDVWGQGTTVTVSS

[0273] SEQ ID NO:3---CD7 VH amino acid sequence;

[0274] EVQLVESGGGLVKPGGSLRLSCAASGLTFSSYAMSWVRQAPEKRLEWVSSISSGGF

[0275] TYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDEVRGYLDVWGQG

[0276] TTVTVSS

[0277] SEQ ID NO:4---CD7 VL amino acid sequence;

[0278] DIQMTQSPSSSLSASVGDRVTITCSASQGISNYLNWYQQKPDKTVKLLIYYTSSLHSG

[0279] VPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKLEIK

[0280] SEQ ID NO:5---CD7 VH CDR1 amino acid sequence;

[0281] SYAMS

[0282] SEQ ID NO:6---CD7 VH CDR2 amino acid sequence;

[0283] SISSGGFTYYPDSVKG

[0284] SEQ ID NO:7---CD7 VH CDR3 amino acid sequence;

[0285] DEVRGYLDV

[0286] SEQ ID NO:8---CD7 VL CDR1 amino acid sequence;

[0287] SASQGISNYLN

[0288] SEQ ID NO:9---CD7 VL CDR2 amino acid sequence;

[0289] YTSSLHS

[0290] SEQ ID NO:10---CD7 VL CDR3 amino acid sequence;

[0291] QQYSKLPYT

[0292] SEQ ID NO:11---Amino acid sequence of the hinge region derived from CD8α;

[0293] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0294] SEQ ID NO:12---Transmembrane amino acid sequence derived from CD8α;

[0295] IYIWAPLAGTCGVLLLSLVITLYC

[0296] SEQ ID NO:13---Amino acid sequence derived from the co-stimulatory signal transduction region of 4-1BB;

[0297] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0298] SEQ ID NO:14---Amino acid sequence derived from the co-stimulatory signal transduction region of CD28;

[0299] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0300] SEQ ID NO:15---Amino acid sequence derived from the intracellular signal transduction region of CD3ζ;

[0301] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP

[0302] QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ

[0303] ALPPR

[0304] SEQ ID NO:16---The intracellular domain contains an amino acid sequence derived from the intracellular region of the CD7 protein;

[0305] RTQIKKLCSWRDKNSAACVVYEDMSHSRCNTLSSPNQYQ

[0306] SEQ ID NO:17---Amino acid sequence of intracellular domain 4-1BB-CD3ζ;

[0307] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQ

[0308] QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM

[0309] AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0310] SEQ ID NO:18---Amino acid sequence of intracellular domain CD28-CD3ζ;

[0311] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQ

[0312] QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM

[0313] AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0314] SEQ ID NO:19---Amino acid sequence of intracellular domain 4-1BB-CD3ζ-γc;

[0315] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQ

[0316] QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM

[0317] AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRERTMPRIPTL

[0318] KNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGAS

[0319] PCNQHSPYWAPPCYTLKPET

[0320] SEQ ID NO:20---Amino acid sequence of the intracellular domain CD28-CD3ζ-γc;

[0321] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQ

[0322] QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM

[0323] AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRERTMPRIPTL

[0324] KNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGAS

[0325] PCNQHSPYWAPPCYTLKPET

[0326] SEQ ID NO:21---Amino acid sequence of intracellular domain CD7-4-1BB-CD3ζ;

[0327] RTQIKKLCSWRDKNSAACVVYEDMSHSRCNTLSSPNQYQKRGRKKLLYIFKQPFM

[0328] RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRR

[0329] EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG

[0330] KGHDGLYQGLSTATKDTYDALHMQALPPR

[0331] SEQ ID NO:22---Amino acid sequence of intracellular domain 4-1BB-CD7-CD3ζ;

[0332] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRTQIKKLCSWRDKN

[0333] SAACVVYEDMSHSRCNTLSSPNQYQRVKFSRSADAPAYQQGQNQLYNELNLGRR

[0334] EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG

[0335] KGHDGLYQGLSTATKDTYDALHMQALPPR

[0336] SEQ ID NO:23---Amino acid sequence of intracellular domain CD7-CD28-CD3ζ;

[0337] RTQIKKLCSWRDKNSAACVVYEDMSHSRCNTLSSPNQYQRSKRSRLLHSDYMNM

[0338] TPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRR

[0339] EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG

[0340] KGHDGLYQGLSTATKDTYDALHMQALPPR

[0341] SEQ ID NO:24---Amino acid sequence of intracellular domain CD28 - CD7 - CD3ζ;

[0342] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRTQIKKLCSWRDKN

[0343] SAACVVYEDMSHSRCNTLSSPNQYQRVKFSRSADAPAYQQGQNQLYNELNLGRR

[0344] EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG

[0345] KGHDGLYQGLSTATKDTYDALHMQALPPR

[0346] SEQ ID NO:25---TRAC chRDNA;

[0347] AGAGTCUCTCAGCUGGUACA

[0348] SEQ ID NO:26---CD7 sgRNA;

[0349] CGGAACCGUCUGUCCGUAGU

[0350] SEQ ID NO:27---Positive control CAR construct 1 group template dsDNA (4 - 1BB - CD3ζ);

[0351] CACCGCCTCCAGGAAGCCCTCTCTGAGCTCTGAGCGCCTGCGGTCTCCTGT

[0352] GTGCTGCTCTCTGTGGGGTCCTGTAGACCCAGAGAGGCTCAGCTGCACTC

[0353] GCCCGGCTGGGAGAGCTGGGTGTGGGGAACATGGCCGGGCCTCCGAGGC

[0354] TCCTGCTGCTGCCCCTGCTTCTGGCGCTGGCTCGCGGCCTGCCTGGGGCC

[0355] CTGGCTGCCCAAGGTAAGAGCTTCCCAGGCTCTCCATGGCCACAGCTCCA

[0356] GAGCTCTCCCTGCCCCATGAGCTCAGAGCCCCCAGTCTGAGCCACAGCAC

[0357] AGCCCCCAGGAAGCGGGTGGGGTGCTGAGCGGCCTCCAGTGTCTGAGGAC

[0358] TCATTTAAGAGAAGGAAAAAGGGTGGACCCCGGTGGGGAGTGGCCGGGGC

[0359] TGTCCAGGCAGGGCCGCTGCTTTGGGAGGAAGAAGCCCACAGTCTCGGAA

[0360] CACGAGGACAGCACCTCCCCCAACACCACAGCCGGTGCCCAGATCTGCTC

[0361] CATGCCCCGTAAGGCACCGTGTCTTTGGCGACATGTCAGCCCTGGGCTGT

[0362] CTCAGGGCCCCACCATCCCCACCACTGTCCCCTGCAGGGAGGACATTCTCT

[0363] GTCCTTCTGGCCAGACTGATGGTGACAGCCCAGGTCCTCCCCAGAGGTGC

[0364] AGCAGTCTCCCCACTGCACGACTGTCCCCGTGGGAGCCTCCGTCAACATCA

[0365] CCTGCTCCACCAGCGGGGGCCTGCGTGGGATCTACCTGAGGCAGCTCGGG

[0366] CCACAGCCCCAAGACATCATTTACTACGAGGACGGGGTGGTGCCCACCCA

[0367] TAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCC

[0368] CCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCTACTCA

[0369] GACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGG

[0370] CACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGC

[0371] TGGCAACTAGAAGGCACAGTTAGCGAGGGGGCAGGGCCTGCATGTGAAGG

[0372] GCGTCGTAGGTGTCCTTGGTGGCTGTACTGAGACCCTGGTAAAGGCCATC

[0373] GTGCCCCTTGCCCCTCCGGCGCTCGCCTTTCATCCCAATCTCACTGTAGGC

[0374] CTCCGCCATCTTATCTTTCTGCAGTTCATTGTACAGGCCTTCCTGAGGGTT

[0375] CTTCCTTCTCGGCTTTCCCCCCATCTCAGGGTCCCGGCCACGTCTCTTGTC

[0376] CAAAACATCGTACTCCTCTCTTCGTCCTAGATTGAGCTCGTTATAGAGCTG

[0377] GTTCTGGCCCTGCTGGTACGCGGGGGCGTCTGCGCTCCTGCTGAACTTCA

[0378] CTCTCAGTTCACATCCTCCTTCTTCTTCTTCTGGAAATCGGCAGCTACAGC

[0379] CATCTTCCTCTTGAGTAGTTTGTACTGGTCTCATAAATGGTTGTTTGAATAT

[0380] ATACAGGAGTTTCTTTCTGCCCCGTTTGCAGTAAAGGGTGATAACCAGTGA

[0381] CAGGAGAAGGACCCCACAAGTCCCGGCCAAGGGCGCCCAGATGTAGATAT

[0382] CACAGGCGAAGTCCAGCCCCCTCGTGTGCACTGCGCCCCCCGCCGCTGGC

[0383] CGGCACGCCTCTGGGCGCAGGGACAGGGGCTGCGACGCGATGGTGGGCG

[0384] CCGGTGTTGGTGGTCGCGGCGCTGGCGTCGTGGTGCTGCTCACGGTCACG

[0385] GTGGTGCCTTGGCCCCACACGTCCAGGTAGCCTCTCACCTCGTCTCTAGCG

[0386] CAGTAGTACACGGCGGTGTCCTCGGCTCTCAGGCTGTTCATCTGCAGGTAC

[0387] AGGCTGTTCTTGGCGTTGTCTCTGCTGATGGTGAATCTGCCCTTCACGCTG

[0388] TCGGGGTAGTAGGTGAAGCCGCCGCTGCTGATGCTGCTCACCCACTCCAG

[0389] TCTCTTCTCGGGGGCTTGTCTCACCCAGCTCATGGCGTAGCTGCTGAAGGT

[0390] CAGGCCGGAGGCGGCGCAGCTCAGTCTCAGGCTGCCGCCAGGCTTCACGA

[0391] GGCCCCCGCCGGACTCCACCAGTTGGACCTCGCTGCCCCCGCCCCCGGAC

[0392] CCGCCCCCGCCGCTCCCGCCCCCGCCCTTAATCTCGAGTTTGGTCCCGCCC

[0393] CCAAATGTGTAGGGCAGCTTGCTGTACTGCTGACAGTAGTAGGTGGCGAA

[0394] GTCCTCAGGCTGCAGGCTGCTGATGGTCAGGGTGTAGTCGGTGCCGCTGC

[0395] CGCTGCCGCTGAATCTGCTAGGCACGCCGCTGTGCAGGCTGCTTGTGTAG

[0396] TAGATCAGCAGCTTCACGGTCTTGTCGGGCTTCTGCTGATACCAGTTCAGG

[0397] TAGTTGCTGATGCCTTGGGAGGCGCTGCAGGTGATGGTCACTCTGTCGCC

[0398] CACGGAGGCGCTCAGGCTGCTAGGGCTCTGTGTCATCTGAATGTCCGGCC

[0399] TGGCGGCGTGGAGCAGCAAGGCCAGCGGCAGGAGCAAGGCGGTCACTGG

[0400] TAAGGCCATGGTGGCAGCGCTCTAGAACCGGTCCTGTGTTCTGGCGGCAA

[0401] ACCCGTTGCGAAAAAGAACGTTCACGGCGACTACTGCACTTATATACGGTT

[0402] CTCCCCCACCCTCGGGAAAAAGGCGGAGCCAGTACACGACATCACTTTCC

[0403] CAGTTTACCCCGCGCCACCTTCTCTAGGCACCCGTTCAATTGCCGACCCCT

[0404] CCCCCCAACTTCTCGGGGACTGTGGGCGATGTGCGCTCTGCCCACGGACA

[0405] GACGGTTCCGGGGCCGCATCGACTTCTCAGGGTCCCAGGACAACCTGACT

[0406] ATCACCATGCACCGCCTGCAGCTGTCGGACACTGGCACCTACACCTGCCA

[0407] GGCCATCACGGAGGTCAATGTCTACGGCTCCGGCACCCTGGTCCTGGTGA

[0408] CAGGTAGGGAATGTGCCCATCCCAGACCCCCCTCCCAACCCCAGCTGCTG

[0409] GCCAGGCTCTGCTCCCCCAGCCCTTGTCGTGGGACCCTCCCTCCTACATGT

[0410] GCCTGAACTGTTCCAGCTCCCAGCCCACTGCCCCCAGCAGCCTCCTAGATA

[0411] GCTGCCCCTCCTCCCCTCCACAGCCTTTCCCTGCCCCGAATCCCAAACCCC

[0412] GGGGGCTCTAACAGGTTCTCCACCGGGAGAATCCCTTCCTTCTTTTTTCCT

[0413] TCTCAGAGGAACAGTCCCAAGGATGGCACAGATGCTCGGACGCCCCACCA

[0414] AGGGCCTCTGCCCTCCCTGCCCCACCGACAGGCTCCGCCCTCCCTGACCC

[0415] GCAGACAGCCTCTGCCCTCCCTGACCCGCCAGCAGCCTCTGCCCTCCCTGC

[0416] GGCCCTGGCGGTGATCTCCTTCCTCCTCGGGCTGGGCCTGGGGGTGGCGT

[0417] GTGTGCTGGCGAGGACACAGGTCAGTGTGAGCCCCAGCTGCCACCTGCAC

[0418] CCCAAAGATTGTTCCCTCTCCTGAGAGCAGCGTGGGGGGCGCCAAAGCCC

[0419] CAGAGGAAAACCCTGGTACCCGCCGCCTCCTCTGCAGGCTCTGGAACTCT

[0420] CAGGGTGGAAGTGGCCTCGGCGGGGAGGGAGGGGGACC

[0421] SEQ ID NO:28 - Positive control CAR structure group 2 template dsDNA (CD28 - CD3ζ);

[0422] CACCGCCTCCAGGAAGCCCTCTCTGAGCTCTGAGCGCCTGCGGTCTCCTGT

[0423] GTGCTGCTCTCTGTGGGGTCCTGTAGACCCAGAGAGGCTCAGCTGCACTC

[0424] GCCCGGCTGGGAGAGCTGGGTGTGGGGAACATGGCCGGGCCTCCGAGGC

[0425] TCCTGCTGCTGCCCCTGCTTCTGGCGCTGGCTCGCGGCCTGCCTGGGGCC

[0426] CTGGCTGCCCAAGGTAAGAGCTTCCCAGGCTCTCCATGGCCACAGCTCCA

[0427] GAGCTCTCCCTGCCCCATGAGCTCAGAGCCCCCAGTCTGAGCCACAGCAC

[0428] AGCCCCCAGGAAGCGGGTGGGGTGCTGAGCGGCCTCCAGTGTCTGAGGAC

[0429] TCATTTAAGAGAAGGAAAAAGGGTGGACCCCGGTGGGGAGTGGCCGGGGC

[0430] TGTCCAGGCAGGGCCGCTGCTTTGGGAGGAAGAAGCCCACAGTCTCGGAA

[0431] CACGAGGACAGCACCTCCCCCAACACCACAGCCGGTGCCCAGATCTGCTC

[0432] CATGCCCCGTAAGGCACCGTGTCTTTGGCGACATGTCAGCCCTGGGCTGT

[0433] CTCAGGGCCCCACCATCCCCACCACTGTCCCCTGCAGGGAGGACATTCTCT

[0434] GTCCTTCTGGCCAGACTGATGGTGACAGCCCAGGTCCTCCCCAGAGGTGC

[0435] AGCAGTCTCCCCACTGCACGACTGTCCCCGTGGGAGCCTCCGTCAACATCA

[0436] CCTGCTCCACCAGCGGGGGCCTGCGTGGGATCTACCTGAGGCAGCTCGGG

[0437] CCACAGCCCCAAGACATCATTTACTACGAGGACGGGGTGGTGCCCACCCA

[0438] TAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCC

[0439] CCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCTACTCA

[0440] GACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGG

[0441] CACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGC

[0442] TGGCAACTAGAAGGCACAGTTAGCGAGGGGGCAGGGCCTGCATGTGAAGG

[0443] GCGTCGTAGGTGTCCTTGGTGGCTGTACTGAGACCCTGGTAAAGGCCATC

[0444] GTGCCCCTTGCCCCTCCGGCGCTCGCCTTTCATCCCAATCTCACTGTAGGC

[0445] CTCCGCCATCTTATCTTTCTGCAGTTCATTGTACAGGCCTTCCTGAGGGTT

[0446] CTTCCTTCTCGGCTTTCCCCCCATCTCAGGGTCCCGGCCACGTCTCTTGTC

[0447] CAAAACATCGTACTCCTCTCTTCGTCCTAGATTGAGCTCGTTATAGAGCTG

[0448] GTTCTGGCCCTGCTGGTACGCGGGGGCGTCTGCGCTCCTGCTGAACTTCA

[0449] CTCTGGAGCGATAGGCTGCGAAGTCGCGTGGTGGGGCATAGGGCTGGTAA

[0450] TGCTTGCGGGTGGGCCCGGGGCGGCGGGGAGTCATGTTCATGTAGTCACT

[0451] GTGCAGGAGCCTGCTCCTCTTACTCCTGCAGTAAAGGGTGATAACCAGTGA

[0452] CAGGAGAAGGACCCCACAAGTCCCGGCCAAGGGCGCCCAGATGTAGATAT

[0453] CACAGGCGAAGTCCAGCCCCCTCGTGTGCACTGCGCCCCCCGCCGCTGGC

[0454] CGGCACGCCTCTGGGCGCAGGGACAGGGGCTGCGACGCGATGGTGGGCG

[0455] CCGGTGTTGGTGGTCGCGGCGCTGGCGTCGTGGTGCTGCTCACGGTCACG

[0456] GTGGTGCCTTGGCCCCACACGTCCAGGTAGCCTCTCACCTCGTCTCTAGCG

[0457] CAGTAGTACACGGCGGTGTCCTCGGCTCTCAGGCTGTTCATCTGCAGGTAC

[0458] AGGCTGTTCTTGGCGTTGTCTCTGCTGATGGTGAATCTGCCCTTCACGCTG

[0459] TCGGGGTAGTAGGTGAAGCCGCCGCTGCTGATGCTGCTCACCCACTCCAG

[0460] TCTCTTCTCGGGGGCTTGTCTCACCCAGCTCATGGCGTAGCTGCTGAAGGT

[0461] CAGGCCGGAGGCGGCGCAGCTCAGTCTCAGGCTGCCGCCAGGCTTCACGA

[0462] GGCCCCCGCCGGACTCCACCAGTTGGACCTCGCTGCCCCCGCCCCCGGAC

[0463] CCGCCCCCGCCGCTCCCGCCCCCGCCCTTAATCTCGAGTTTGGTCCCGCCC

[0464] CCAAATGTGTAGGGCAGCTTGCTGTACTGCTGACAGTAGTAGGTGGCGAA

[0465] GTCCTCAGGCTGCAGGCTGCTGATGGTCAGGGTGTAGTCGGTGCCGCTGC

[0466] CGCTGCCGCTGAATCTGCTAGGCACGCCGCTGTGCAGGCTGCTTGTGTAG

[0467] TAGATCAGCAGCTTCACGGTCTTGTCGGGCTTCTGCTGATACCAGTTCAGG

[0468] TAGTTGCTGATGCCTTGGGAGGCGCTGCAGGTGATGGTCACTCTGTCGCC

[0469] CACGGAGGCGCTCAGGCTGCTAGGGCTCTGTGTCATCTGAATGTCCGGCC

[0470] TGGCGGCGTGGAGCAGCAAGGCCAGCGGCAGGAGCAAGGCGGTCACTGG

[0471] TAAGGCCATGGTGGCAGCGCTCTAGAACCGGTCCTGTGTTCTGGCGGCAA

[0472] ACCCGTTGCGAAAAAGAACGTTCACGGCGACTACTGCACTTATATACGGTT

[0473] CTCCCCCACCCTCGGGAAAAAGGCGGAGCCAGTACACGACATCACTTTCC

[0474] CAGTTTACCCCGCGCCACCTTCTCTAGGCACCCGTTCAATTGCCGACCCCT

[0475] CCCCCCAACTTCTCGGGGACTGTGGGCGATGTGCGCTCTGCCCACGGACA

[0476] GACGGTTCCGGGGCCGCATCGACTTCTCAGGGTCCCAGGACAACCTGACT

[0477] ATCACCATGCACCGCCTGCAGCTGTCGGACACTGGCACCTACACCTGCCA

[0478] GGCCATCACGGAGGTCAATGTCTACGGCTCCGGCACCCTGGTCCTGGTGA

[0479] CAGGTAGGGAATGTGCCCATCCCAGACCCCCCTCCCAACCCCAGCTGCTG

[0480] GCCAGGCTCTGCTCCCCCAGCCCTTGTCGTGGGACCCTCCCTCCTACATGT

[0481] GCCTGAACTGTTCCAGCTCCCAGCCCACTGCCCCCAGCAGCCTCCTAGATA

[0482] GCTGCCCCTCCTCCCCTCCACAGCCTTTCCCTGCCCCGAATCCCAAACCCC

[0483] GGGGGCTCTAACAGGTTCTCCACCGGGAGAATCCCTTCCTTCTTTTTTCCT

[0484] TCTCAGAGGAACAGTCCCAAGGATGGCACAGATGCTCGGACGCCCCACCA

[0485] AGGGCCTCTGCCCTCCCTGCCCCACCGACAGGCTCCGCCCTCCCTGACCC

[0486] GCAGACAGCCTCTGCCCTCCCTGACCCGCCAGCAGCCTCTGCCCTCCCTGC

[0487] GGCCCTGGCGGTGATCTCCTTCCTCCTCGGGCTGGGCCTGGGGGTGGCGT

[0488] GTGTGCTGGCGAGGACACAGGTCAGTGTGAGCCCCAGCTGCCACCTGCAC

[0489] CCCAAAGATTGTTCCCTCTCCTGAGAGCAGCGTGGGGGGCGCCAAAGCCC

[0490] CAGAGGAAAACCCTGGTACCCGCCGCCTCCTCTGCAGGCTCTGGAACTCT

[0491] CAGGGTGGAAGTGGCCTCGGCGGGGAGGGAGGGGGACC

[0492]

[0493] ACCCCGGGGGCTCTAACAGGTTCTCCACCGGGAGAATCCCTTCCTTCTTTT

[0494] TTCCTTCTCAGAGGAACAGTCCCAAGGATGGCACAGATGCTCGGACGCCC

[0495] CACCAAGGGCCTCTGCCCTCCCTGCCCCACCGACAGGCTCCGCCCTCCCT

[0496] GACCCGCAGACAGCCTCTGCCCTCCCTGACCCGCCAGCAGCCTCTGCCCT

[0497] CCCTGCGGCCCTGGCGGTGATCTCCTTCCTCCTCGGGCTGGGCCTGGGGG

[0498] TGGCGTGTGTGCTGGCGAGGACACAGGTCAGTGTGAGCCCCAGCTGCCAC

[0499] CTGCACCCCAAAGATTGTTCCCTCTCCTGAGAGCAGCGTGGGGGGCGCCA

[0500] AAGCCCCAGAGGAAAACCCTGGTACCCGCCGCCTCCTCTGCAGGCTCTGG

[0501] AACTCTCAGGGTGGAAGTGGCCTCGGCGGGGAGGGAGGGGGACC

[0502] SEQ ID NO:30---Candidate enhanced CAR construct 2 group template dsDNA (CD28-CD3ζ-γc);

[0503] CACCGCCTCCAGGAAGCCCTCTCTGAGCTCTGAGCGCCTGCGGTCTCCTGT

[0504] GTGCTGCTCTCTGTGGGGTCCTGTAGACCCAGAGAGGCTCAGCTGCACTC

[0505] GCCCGGCTGGGAGAGCTGGGTGTGGGGAACATGGCCGGGCCTCCGAGGC

[0506] TCCTGCTGCTGCCCCTGCTTCTGGCGCTGGCTCGCGGCCTGCCTGGGGCC

[0507] CTGGCTGCCCAAGGTAAGAGCTTCCCAGGCTCTCCATGGCCACAGCTCCA

[0508] GAGCTCTCCCTGCCCCATGAGCTCAGAGCCCCCAGTCTGAGCCACAGCAC

[0509] AGCCCCCAGGAAGCGGGTGGGGTGCTGAGCGGCCTCCAGTGTCTGAGGAC

[0510] TCATTTAAGAGAAGGAAAAAGGGTGGACCCCGGTGGGGAGTGGCCGGGGC

[0511] TGTCCAGGCAGGGCCGCTGCTTTGGGAGGAAGAAGCCCACAGTCTCGGAA

[0512] CACGAGGACAGCACCTCCCCCAACACCACAGCCGGTGCCCAGATCTGCTC

[0513] CATGCCCCGTAAGGCACCGTGTCTTTGGCGACATGTCAGCCCTGGGCTGT

[0514] CTCAGGGCCCCACCATCCCCACCACTGTCCCCTGCAGGGAGGACATTCTCT

[0515] GTCCTTCTGGCCAGACTGATGGTGACAGCCCAGGTCCTCCCCAGAGGTGC

[0516] AGCAGTCTCCCCACTGCACGACTGTCCCCGTGGGAGCCTCCGTCAACATCA

[0517] CCTGCTCCACCAGCGGGGGCCTGCGTGGGATCTACCTGAGGCAGCTCGGG

[0518] CCACAGCCCCAAGACATCATTTACTACGAGGACGGGGTGGTGCCCACCCA

[0519] TAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCC

[0520] CCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCTACTCA

[0521] GACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGG

[0522] CACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGC

[0523] TGGCAACTAGAAGGCACAGTTAGGTTTCAGGCTTTAGGGTGTAACATGGG

[0524] GGGGCCCAGTAGGGGCTATGCTGGTTGCATGGGGAGGCCCCAGGCCCCTC

[0525] CCCAAGGGCCCCTCCTTTTGGGGGAATCTCACTGACGAGGCAGAGTCGTT

[0526] CACTGTAGTCTGGCTGCAGACTCTCAGCCAGTCCCTTAGACACACCACTCC

[0527] AGGCCGAAAAGTTCCCGTGGTATTCAGTAACAAGATCCTCTAGGTTCTTCA

[0528] GGGTGGGAATTCGGGGCATCGTCCGTTCGCGAGGGGGCAGGGCCTGCATG

[0529] TGAAGGGCGTCGTAGGTGTCCTTGGTGGCTGTACTGAGACCCTGGTAAAG

[0530] GCCATCGTGCCCCTTGCCCCTCCGGCGCTCGCCTTTCATCCCAATCTCACT

[0531] GTAGGCCTCCGCCATCTTATCTTTCTGCAGTTCATTGTACAGGCCTTCCTG

[0532] AGGGTTCTTCCTTCTCGGCTTTCCCCCCATCTCAGGGTCCCGGCCACGTCT

[0533] CTTGTCCAAAACATCGTACTCCTCTCTTCGTCCTAGATTGAGCTCGTTATA

[0534] GAGCTGGTTCTGGCCCTGCTGGTACGCGGGGGCGTCTGCGCTCCTGCTGA

[0535] ACTTCACTCTGGAGCGATAGGCTGCGAAGTCGCGTGGTGGGGCATAGGGC

[0536] TGGTAATGCTTGCGGGTGGGCCCGGGGCGGCGGGGAGTCATGTTCATGTA

[0537] GTCACTGTGCAGGAGCCTGCTCCTCTTACTCCTGCAGTAAAGGGTGATAAC

[0538] CAGTGACAGGAGAAGGACCCCACAAGTCCCGGCCAAGGGCGCCCAGATGT

[0539] AGATATCACAGGCGAAGTCCAGCCCCCTCGTGTGCACTGCGCCCCCCGCC

[0540] GCTGGCCGGCACGCCTCTGGGCGCAGGGACAGGGGCTGCGACGCGATGG

[0541] TGGGCGCCGGTGTTGGTGGTCGCGGCGCTGGCGTCGTGGTGCTGCTCACG

[0542] GTCACGGTGGTGCCTTGGCCCCACACGTCCAGGTAGCCTCTCACCTCGTCT

[0543] CTAGCGCAGTAGTACACGGCGGTGTCCTCGGCTCTCAGGCTGTTCATCTGC

[0544] AGGTACAGGCTGTTCTTGGCGTTGTCTCTGCTGATGGTGAATCTGCCCTTC

[0545] ACGCTGTCGGGGTAGTAGGTGAAGCCGCCGCTGCTGATGCTGCTCACCCA

[0546] CTCCAGTCTCTTCTCGGGGGCTTGTCTCACCCAGCTCATGGCGTAGCTGCT

[0547] GAAGGTCAGGCCGGAGGCGGCGCAGCTCAGTCTCAGGCTGCCGCCAGGCT

[0548] TCACGAGGCCCCCGCCGGACTCCACCAGTTGGACCTCGCTGCCCCCGCCC

[0549] CCGGACCCGCCCCCGCCGCTCCCGCCCCCGCCCTTAATCTCGAGTTTGGTC

[0550] CCGCCCCCAAATGTGTAGGGCAGCTTGCTGTACTGCTGACAGTAGTAGGT

[0551] GGCGAAGTCCTCAGGCTGCAGGCTGCTGATGGTCAGGGTGTAGTCGGTGC

[0552] CGCTGCCGCTGCCGCTGAATCTGCTAGGCACGCCGCTGTGCAGGCTGCTT

[0553] GTGTAGTAGATCAGCAGCTTCACGGTCTTGTCGGGCTTCTGCTGATACCAG

[0554] TTCAGGTAGTTGCTGATGCCTTGGGAGGCGCTGCAGGTGATGGTCACTCT

[0555] GTCGCCCACGGAGGCGCTCAGGCTGCTAGGGCTCTGTGTCATCTGAATGT

[0556] CCGGCCTGGCGGCGTGGAGCAGCAAGGCCAGCGGCAGGAGCAAGGCGGT

[0557] CACTGGTAAGGCCATGGTGGCAGCGCTCTAGAACCGGTCCTGTGTTCTGG

[0558] CGGCAAACCCGTTGCGAAAAAGAACGTTCACGGCGACTACTGCACTTATAT

[0559] ACGGTTCTCCCCCACCCTCGGGAAAAAGGCGGAGCCAGTACACGACATCA

[0560] CTTTCCCAGTTTACCCCGCGCCACCTTCTCTAGGCACCCGTTCAATTGCCG

[0561] ACCCCTCCCCCCAACTTCTCGGGGACTGTGGGCGATGTGCGCTCTGCCCA

[0562] CGGACAGACGGTTCCGGGGCCGCATCGACTTCTCAGGGTCCCAGGACAAC

[0563] CTGACTATCACCATGCACCGCCTGCAGCTGTCGGACACTGGCACCTACACC

[0564] TGCCAGGCCATCACGGAGGTCAATGTCTACGGCTCCGGCACCCTGGTCCT

[0565] GGTGACAGGTAGGGAATGTGCCCATCCCAGACCCCCCTCCCAACCCCAGC

[0566] TGCTGGCCAGGCTCTGCTCCCCCAGCCCTTGTCGTGGGACCCTCCCTCCTA

[0567] CATGTGCCTGAACTGTTCCAGCTCCCAGCCCACTGCCCCCAGCAGCCTCCT

[0568] AGATAGCTGCCCCTCCTCCCCTCCACAGCCTTTCCCTGCCCCGAATCCCAA

[0569] ACCCCGGGGGCTCTAACAGGTTCTCCACCGGGAGAATCCCTTCCTTCTTTT

[0570] TTCCTTCTCAGAGGAACAGTCCCAAGGATGGCACAGATGCTCGGACGCCC

[0571] CACCAAGGGCCTCTGCCCTCCCTGCCCCACCGACAGGCTCCGCCCTCCCT

[0572] GACCCGCAGACAGCCTCTGCCCTCCCTGACCCGCCAGCAGCCTCTGCCCT

[0573] CCCTGCGGCCCTGGCGGTGATCTCCTTCCTCCTCGGGCTGGGCCTGGGGG

[0574] TGGCGTGTGTGCTGGCGAGGACACAGGTCAGTGTGAGCCCCAGCTGCCAC

[0575] CTGCACCCCAAAGATTGTTCCCTCTCCTGAGAGCAGCGTGGGGGGCGCCA

[0576] AAGCCCCAGAGGAAAACCCTGGTACCCGCCGCCTCCTCTGCAGGCTCTGG

[0577] AACTCTCAGGGTGGAAGTGGCCTCGGCGGGGAGGGAGGGGGACC

[0578] SEQ ID NO:31 - Candidate enhanced CAR construct group 3 template dsDNA (CD7-4-1BB-CD3ζ);

[0579] CACCGCCTCCAGGAAGCCCTCTCTGAGCTCTGAGCGCCTGCGGTCTCCTGT

[0580] GTGCTGCTCTCTGTGGGGTCCTGTAGACCCAGAGAGGCTCAGCTGCACTC

[0581] GCCCGGCTGGGAGAGCTGGGTGTGGGGAACATGGCCGGGCCTCCGAGGC

[0582] TCCTGCTGCTGCCCCTGCTTCTGGCGCTGGCTCGCGGCCTGCCTGGGGCC

[0583] CTGGCTGCCCAAGGTAAGAGCTTCCCAGGCTCTCCATGGCCACAGCTCCA

[0584] GAGCTCTCCCTGCCCCATGAGCTCAGAGCCCCCAGTCTGAGCCACAGCAC

[0585] AGCCCCCAGGAAGCGGGTGGGGTGCTGAGCGGCCTCCAGTGTCTGAGGAC

[0586] TCATTTAAGAGAAGGAAAAAGGGTGGACCCCGGTGGGGAGTGGCCGGGGC

[0587] TGTCCAGGCAGGGCCGCTGCTTTGGGAGGAAGAAGCCCACAGTCTCGGAA

[0588] CACGAGGACAGCACCTCCCCCAACACCACAGCCGGTGCCCAGATCTGCTC

[0589] CATGCCCCGTAAGGCACCGTGTCTTTGGCGACATGTCAGCCCTGGGCTGT

[0590] CTCAGGGCCCCACCATCCCCACCACTGTCCCCTGCAGGGAGGACATTCTCT

[0591] GTCCTTCTGGCCAGACTGATGGTGACAGCCCAGGTCCTCCCCAGAGGTGC

[0592] AGCAGTCTCCCCACTGCACGACTGTCCCCGTGGGAGCCTCCGTCAACATCA

[0593] CCTGCTCCACCAGCGGGGGCCTGCGTGGGATCTACCTGAGGCAGCTCGGG

[0594] CCACAGCCCCAAGACATCATTTACTACGAGGACGGGGTGGTGCCCACCCA

[0595] TAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCC

[0596] CCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCTACTCA

[0597] GACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGG

[0598] CACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGC

[0599] TGGCAACTAGAAGGCACAGTTAGCGAGGGGGCAGGGCCTGCATGTGAAGG

[0600] GCGTCGTAGGTGTCCTTGGTGGCTGTACTGAGACCCTGGTAAAGGCCATC

[0601] GTGCCCCTTGCCCCTCCGGCGCTCGCCTTTCATCCCAATCTCACTGTAGGC

[0602] CTCCGCCATCTTATCTTTCTGCAGTTCATTGTACAGGCCTTCCTGAGGGTT

[0603] CTTCCTTCTCGGCTTTCCCCCCATCTCAGGGTCCCGGCCACGTCTCTTGTC

[0604] CAAAACATCGTACTCCTCTCTTCGTCCTAGATTGAGCTCGTTATAGAGCTG

[0605] GTTCTGGCCCTGCTGGTACGCGGGGGCGTCTGCGCTCCTGCTGAACTTCA

[0606] CTCTCAGTTCACATCCTCCTTCTTCTTCTTCTGGAAATCGGCAGCTACAGC

[0607] CATCTTCCTCTTGAGTAGTTTGTACTGGTCTCATAAATGGTTGTTTGAATAT

[0608] ATACAGGAGTTTCTTTCTGCCCCGTTTCTGGTACTGGTTGGGGGAGGACAG

[0609] CGTGTTGCAGCGGCTGTGCGACATGTCCTCGTACACCACACATGCCGCCG

[0610] AATTCTTATCCCGCCACGAGCACAGTTTCTTTATCTGTGTCCTGCAGTAAA

[0611] GGGTGATAACCAGTGACAGGAGAAGGACCCCACAAGTCCCGGCCAAGGGC

[0612] GCCCAGATGTAGATATCACAGGCGAAGTCCAGCCCCCTCGTGTGCACTGC

[0613] GCCCCCCGCCGCTGGCCGGCACGCCTCTGGGCGCAGGGACAGGGGCTGC

[0614] GACGCGATGGTGGGCGCCGGTGTTGGTGGTCGCGGCGCTGGCGTCGTGGT

[0615] GCTGCTCACGGTCACGGTGGTGCCTTGGCCCCACACGTCCAGGTAGCCTC

[0616] TCACCTCGTCTCTAGCGCAGTAGTACACGGCGGTGTCCTCGGCTCTCAGGC

[0617] TGTTCATCTGCAGGTACAGGCTGTTCTTGGCGTTGTCTCTGCTGATGGTGA

[0618] ATCTGCCCTTCACGCTGTCGGGGTAGTAGGTGAAGCCGCCGCTGCTGATG

[0619] CTGCTCACCCACTCCAGTCTCTTCTCGGGGGCTTGTCTCACCCAGCTCATG

[0620] GCGTAGCTGCTGAAGGTCAGGCCGGAGGCGGCGCAGCTCAGTCTCAGGCT

[0621] GCCGCCAGGCTTCACGAGGCCCCCGCCGGACTCCACCAGTTGGACCTCGC

[0622] TGCCCCCGCCCCCGGACCCGCCCCCGCCGCTCCCGCCCCCGCCCTTAATC

[0623] TCGAGTTTGGTCCCGCCCCCAAATGTGTAGGGCAGCTTGCTGTACTGCTGA

[0624] CAGTAGTAGGTGGCGAAGTCCTCAGGCTGCAGGCTGCTGATGGTCAGGGT

[0625] GTAGTCGGTGCCGCTGCCGCTGCCGCTGAATCTGCTAGGCACGCCGCTGT

[0626] GCAGGCTGCTTGTGTAGTAGATCAGCAGCTTCACGGTCTTGTCGGGCTTCT

[0627] GCTGATACCAGTTCAGGTAGTTGCTGATGCCTTGGGAGGCGCTGCAGGTG

[0628] ATGGTCACTCTGTCGCCCACGGAGGCGCTCAGGCTGCTAGGGCTCTGTGT

[0629] CATCTGAATGTCCGGCCTGGCGGCGTGGAGCAGCAAGGCCAGCGGCAGGA

[0630] GCAAGGCGGTCACTGGTAAGGCCATGGTGGCAGCGCTCTAGAACCGGTCC

[0631] TGTGTTCTGGCGGCAAACCCGTTGCGAAAAAGAACGTTCACGGCGACTAC

[0632] TGCACTTATATACGGTTCTCCCCCACCCTCGGGAAAAAGGCGGAGCCAGTA

[0633] CACGACATCACTTTCCCAGTTTACCCCGCGCCACCTTCTCTAGGCACCCGT

[0634] TCAATTGCCGACCCCTCCCCCCAACTTCTCGGGGACTGTGGGCGATGTGC

[0635] GCTCTGCCCACGGACAGACGGTTCCGGGGCCGCATCGACTTCTCAGGGTC

[0636] CCAGGACAACCTGACTATCACCATGCACCGCCTGCAGCTGTCGGACACTG

[0637] GCACCTACACCTGCCAGGCCATCACGGAGGTCAATGTCTACGGCTCCGGC

[0638] ACCCTGGTCCTGGTGACAGGTAGGGAATGTGCCCATCCCAGACCCCCCTC

[0639] CCAACCCCAGCTGCTGGCCAGGCTCTGCTCCCCCAGCCCTTGTCGTGGGA

[0640] CCCTCCCTCCTACATGTGCCTGAACTGTTCCAGCTCCCAGCCCACTGCCCC

[0641] CAGCAGCCTCCTAGATAGCTGCCCCTCCTCCCCTCCACAGCCTTTCCCTGC

[0642] CCCGAATCCCAAACCCCGGGGGCTCTAACAGGTTCTCCACCGGGAGAATC

[0643] CCTTCCTTCTTTTTTCCTTCTCAGAGGAACAGTCCCAAGGATGGCACAGAT

[0644] GCTCGGACGCCCCACCAAGGGCCTCTGCCCTCCCTGCCCCACCGACAGGC

[0645] TCCGCCCTCCCTGACCCGCAGACAGCCTCTGCCCTCCCTGACCCGCCAGCA

[0646] GCCTCTGCCCTCCCTGCGGCCCTGGCGGTGATCTCCTTCCTCCTCGGGCTG

[0647] GGCCTGGGGGTGGCGTGTGTGCTGGCGAGGACACAGGTCAGTGTGAGCCC

[0648] CAGCTGCCACCTGCACCCCAAAGATTGTTCCCTCTCCTGAGAGCAGCGTGG

[0649] GGGGCGCCAAAGCCCCAGAGGAAAACCCTGGTACCCGCCGCCTCCTCTGC

[0650] AGGCTCTGGAACTCTCAGGGTGGAAGTGGCCTCGGCGGGGAGGGAGGGG

[0651] GACC

[0652] SEQ ID NO:32 - Candidate enhanced CAR structure 4 - group template dsDNA (4 - 1BB - CD7 - CD3ζ);

[0653] CACCGCCTCCAGGAAGCCCTCTCTGAGCTCTGAGCGCCTGCGGTCTCCTGT

[0654] GTGCTGCTCTCTGTGGGGTCCTGTAGACCCAGAGAGGCTCAGCTGCACTC

[0655] GCCCGGCTGGGAGAGCTGGGTGTGGGGAACATGGCCGGGCCTCCGAGGC

[0656] TCCTGCTGCTGCCCCTGCTTCTGGCGCTGGCTCGCGGCCTGCCTGGGGCC

[0657] CTGGCTGCCCAAGGTAAGAGCTTCCCAGGCTCTCCATGGCCACAGCTCCA

[0658] GAGCTCTCCCTGCCCCATGAGCTCAGAGCCCCCAGTCTGAGCCACAGCAC

[0659] AGCCCCCAGGAAGCGGGTGGGGTGCTGAGCGGCCTCCAGTGTCTGAGGAC

[0660] TCATTTAAGAGAAGGAAAAAGGGTGGACCCCGGTGGGGAGTGGCCGGGGC

[0661] TGTCCAGGCAGGGCCGCTGCTTTGGGAGGAAGAAGCCCACAGTCTCGGAA

[0662] CACGAGGACAGCACCTCCCCCAACACCACAGCCGGTGCCCAGATCTGCTC

[0663] CATGCCCCGTAAGGCACCGTGTCTTTGGCGACATGTCAGCCCTGGGCTGT

[0664] CTCAGGGCCCCACCATCCCCACCACTGTCCCCTGCAGGGAGGACATTCTCT

[0665] GTCCTTCTGGCCAGACTGATGGTGACAGCCCAGGTCCTCCCCAGAGGTGC

[0666] AGCAGTCTCCCCACTGCACGACTGTCCCCGTGGGAGCCTCCGTCAACATCA

[0667] CCTGCTCCACCAGCGGGGGCCTGCGTGGGATCTACCTGAGGCAGCTCGGG

[0668] CCACAGCCCCAAGACATCATTTACTACGAGGACGGGGTGGTGCCCACCCA

[0669] TAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCC

[0670] CCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCTACTCA

[0671] GACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGG

[0672] CACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGC

[0673] TGGCAACTAGAAGGCACAGTTAGCGAGGGGGCAGGGCCTGCATGTGAAGG

[0674] GCGTCGTAGGTGTCCTTGGTGGCTGTACTGAGACCCTGGTAAAGGCCATC

[0675] GTGCCCCTTGCCCCTCCGGCGCTCGCCTTTCATCCCAATCTCACTGTAGGC

[0676] CTCCGCCATCTTATCTTTCTGCAGTTCATTGTACAGGCCTTCCTGAGGGTT

[0677] CTTCCTTCTCGGCTTTCCCCCCATCTCAGGGTCCCGGCCACGTCTCTTGTC

[0678] CAAAACATCGTACTCCTCTCTTCGTCCTAGATTGAGCTCGTTATAGAGCTG

[0679] GTTCTGGCCCTGCTGGTACGCGGGGGCGTCTGCGCTCCTGCTGAACTTCA

[0680] CTCTCTGGTACTGGTTGGGGGAGGACAGCGTGTTGCAGCGGCTGTGCGAC

[0681] ATGTCCTCGTACACCACACATGCCGCCGAATTCTTATCCCGCCACGAGCAC

[0682] AGTTTCTTTATCTGTGTCCTCAGTTCACATCCTCCTTCTTCTTCTTCTGGAA

[0683] ATCGGCAGCTACAGCCATCTTCCTCTTGAGTAGTTTGTACTGGTCTCATAA

[0684] ATGGTTGTTTGAATATATACAGGAGTTTCTTTCTGCCCCGTTTGCAGTAAA

[0685] GGGTGATAACCAGTGACAGGAGAAGGACCCCACAAGTCCCGGCCAAGGGC

[0686] GCCCAGATGTAGATATCACAGGCGAAGTCCAGCCCCCTCGTGTGCACTGC

[0687] GCCCCCCGCCGCTGGCCGGCACGCCTCTGGGCGCAGGGACAGGGGCTGC

[0688] GACGCGATGGTGGGCGCCGGTGTTGGTGGTCGCGGCGCTGGCGTCGTGGT

[0689] GCTGCTCACGGTCACGGTGGTGCCTTGGCCCCACACGTCCAGGTAGCCTC

[0690] TCACCTCGTCTCTAGCGCAGTAGTACACGGCGGTGTCCTCGGCTCTCAGGC

[0691] TGTTCATCTGCAGGTACAGGCTGTTCTTGGCGTTGTCTCTGCTGATGGTGA

[0692] ATCTGCCCTTCACGCTGTCGGGGTAGTAGGTGAAGCCGCCGCTGCTGATG

[0693] CTGCTCACCCACTCCAGTCTCTTCTCGGGGGCTTGTCTCACCCAGCTCATG

[0694] GCGTAGCTGCTGAAGGTCAGGCCGGAGGCGGCGCAGCTCAGTCTCAGGCT

[0695] GCCGCCAGGCTTCACGAGGCCCCCGCCGGACTCCACCAGTTGGACCTCGC

[0696] TGCCCCCGCCCCCGGACCCGCCCCCGCCGCTCCCGCCCCCGCCCTTAATC

[0697] TCGAGTTTGGTCCCGCCCCCAAATGTGTAGGGCAGCTTGCTGTACTGCTGA

[0698] CAGTAGTAGGTGGCGAAGTCCTCAGGCTGCAGGCTGCTGATGGTCAGGGT

[0699] GTAGTCGGTGCCGCTGCCGCTGCCGCTGAATCTGCTAGGCACGCCGCTGT

[0700] GCAGGCTGCTTGTGTAGTAGATCAGCAGCTTCACGGTCTTGTCGGGCTTCT

[0701] GCTGATACCAGTTCAGGTAGTTGCTGATGCCTTGGGAGGCGCTGCAGGTG

[0702] ATGGTCACTCTGTCGCCCACGGAGGCGCTCAGGCTGCTAGGGCTCTGTGT

[0703] CATCTGAATGTCCGGCCTGGCGGCGTGGAGCAGCAAGGCCAGCGGCAGGA

[0704] GCAAGGCGGTCACTGGTAAGGCCATGGTGGCAGCGCTCTAGAACCGGTCC

[0705] TGTGTTCTGGCGGCAAACCCGTTGCGAAAAAGAACGTTCACGGCGACTAC

[0706] TGCACTTATATACGGTTCTCCCCCACCCTCGGGAAAAAGGCGGAGCCAGTA

[0707] CACGACATCACTTTCCCAGTTTACCCCGCGCCACCTTCTCTAGGCACCCGT

[0708] TCAATTGCCGACCCCTCCCCCCAACTTCTCGGGGACTGTGGGCGATGTGC

[0709] GCTCTGCCCACGGACAGACGGTTCCGGGGCCGCATCGACTTCTCAGGGTC

[0710] CCAGGACAACCTGACTATCACCATGCACCGCCTGCAGCTGTCGGACACTG

[0711] GCACCTACACCTGCCAGGCCATCACGGAGGTCAATGTCTACGGCTCCGGC

[0712] ACCCTGGTCCTGGTGACAGGTAGGGAATGTGCCCATCCCAGACCCCCCTC

[0713] CCAACCCCAGCTGCTGGCCAGGCTCTGCTCCCCCAGCCCTTGTCGTGGGA

[0714] CCCTCCCTCCTACATGTGCCTGAACTGTTCCAGCTCCCAGCCCACTGCCCC

[0715] CAGCAGCCTCCTAGATAGCTGCCCCTCCTCCCCTCCACAGCCTTTCCCTGC

[0716] CCCGAATCCCAAACCCCGGGGGCTCTAACAGGTTCTCCACCGGGAGAATC

[0717] CCTTCCTTCTTTTTTCCTTCTCAGAGGAACAGTCCCAAGGATGGCACAGAT

[0718] GCTCGGACGCCCCACCAAGGGCCTCTGCCCTCCCTGCCCCACCGACAGGC

[0719] TCCGCCCTCCCTGACCCGCAGACAGCCTCTGCCCTCCCTGACCCGCCAGCA

[0720] GCCTCTGCCCTCCCTGCGGCCCTGGCGGTGATCTCCTTCCTCCTCGGGCTG

[0721] GGCCTGGGGGTGGCGTGTGTGCTGGCGAGGACACAGGTCAGTGTGAGCCC

[0722] CAGCTGCCACCTGCACCCCAAAGATTGTTCCCTCTCCTGAGAGCAGCGTGG

[0723] GGGGCGCCAAAGCCCCAGAGGAAAACCCTGGTACCCGCCGCCTCCTCTGC

[0724] AGGCTCTGGAACTCTCAGGGTGGAAGTGGCCTCGGCGGGGAGGGAGGGG

[0725] GACC

[0726] SEQ ID NO:33 - Candidate enhanced CAR structure 5 - group template dsDNA (CD7 - CD28 - CD3ζ);

[0727] CACCGCCTCCAGGAAGCCCTCTCTGAGCTCTGAGCGCCTGCGGTCTCCTGT

[0728] GTGCTGCTCTCTGTGGGGTCCTGTAGACCCAGAGAGGCTCAGCTGCACTC

[0729] GCCCGGCTGGGAGAGCTGGGTGTGGGGAACATGGCCGGGCCTCCGAGGC

[0730] TCCTGCTGCTGCCCCTGCTTCTGGCGCTGGCTCGCGGCCTGCCTGGGGCC

[0731] CTGGCTGCCCAAGGTAAGAGCTTCCCAGGCTCTCCATGGCCACAGCTCCA

[0732] GAGCTCTCCCTGCCCCATGAGCTCAGAGCCCCCAGTCTGAGCCACAGCAC

[0733] AGCCCCCAGGAAGCGGGTGGGGTGCTGAGCGGCCTCCAGTGTCTGAGGAC

[0734] TCATTTAAGAGAAGGAAAAAGGGTGGACCCCGGTGGGGAGTGGCCGGGGC

[0735] TGTCCAGGCAGGGCCGCTGCTTTGGGAGGAAGAAGCCCACAGTCTCGGAA

[0736] CACGAGGACAGCACCTCCCCCAACACCACAGCCGGTGCCCAGATCTGCTC

[0737] CATGCCCCGTAAGGCACCGTGTCTTTGGCGACATGTCAGCCCTGGGCTGT

[0738] CTCAGGGCCCCACCATCCCCACCACTGTCCCCTGCAGGGAGGACATTCTCT

[0739] GTCCTTCTGGCCAGACTGATGGTGACAGCCCAGGTCCTCCCCAGAGGTGC

[0740] AGCAGTCTCCCCACTGCACGACTGTCCCCGTGGGAGCCTCCGTCAACATCA

[0741] CCTGCTCCACCAGCGGGGGCCTGCGTGGGATCTACCTGAGGCAGCTCGGG

[0742] CCACAGCCCCAAGACATCATTTACTACGAGGACGGGGTGGTGCCCACCCA

[0743] TAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCC

[0744] CCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCTACTCA

[0745] GACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGG

[0746] CACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGC

[0747] TGGCAACTAGAAGGCACAGTTAGCGAGGGGGCAGGGCCTGCATGTGAAGG

[0748] GCGTCGTAGGTGTCCTTGGTGGCTGTACTGAGACCCTGGTAAAGGCCATC

[0749] GTGCCCCTTGCCCCTCCGGCGCTCGCCTTTCATCCCAATCTCACTGTAGGC

[0750] CTCCGCCATCTTATCTTTCTGCAGTTCATTGTACAGGCCTTCCTGAGGGTT

[0751] CTTCCTTCTCGGCTTTCCCCCCATCTCAGGGTCCCGGCCACGTCTCTTGTC

[0752] CAAAACATCGTACTCCTCTCTTCGTCCTAGATTGAGCTCGTTATAGAGCTG

[0753] GTTCTGGCCCTGCTGGTACGCGGGGGCGTCTGCGCTCCTGCTGAACTTCA

[0754] CTCTGGAGCGATAGGCTGCGAAGTCGCGTGGTGGGGCATAGGGCTGGTAA

[0755] TGCTTGCGGGTGGGCCCGGGGCGGCGGGGAGTCATGTTCATGTAGTCACT

[0756] GTGCAGGAGCCTGCTCCTCTTACTCCTCTGGTACTGGTTGGGGGAGGACA

[0757] GCGTGTTGCAGCGGCTGTGCGACATGTCCTCGTACACCACACATGCCGCC

[0758] GAATTCTTATCCCGCCACGAGCACAGTTTCTTTATCTGTGTCCTGCAGTAA

[0759] AGGGTGATAACCAGTGACAGGAGAAGGACCCCACAAGTCCCGGCCAAGGG

[0760] CGCCCAGATGTAGATATCACAGGCGAAGTCCAGCCCCCTCGTGTGCACTG

[0761] CGCCCCCCGCCGCTGGCCGGCACGCCTCTGGGCGCAGGGACAGGGGCTGC

[0762] GACGCGATGGTGGGCGCCGGTGTTGGTGGTCGCGGCGCTGGCGTCGTGGT

[0763] GCTGCTCACGGTCACGGTGGTGCCTTGGCCCCACACGTCCAGGTAGCCTC

[0764] TCACCTCGTCTCTAGCGCAGTAGTACACGGCGGTGTCCTCGGCTCTCAGGC

[0765] TGTTCATCTGCAGGTACAGGCTGTTCTTGGCGTTGTCTCTGCTGATGGTGA

[0766] ATCTGCCCTTCACGCTGTCGGGGTAGTAGGTGAAGCCGCCGCTGCTGATG

[0767] CTGCTCACCCACTCCAGTCTCTTCTCGGGGGCTTGTCTCACCCAGCTCATG

[0768] GCGTAGCTGCTGAAGGTCAGGCCGGAGGCGGCGCAGCTCAGTCTCAGGCT

[0769] GCCGCCAGGCTTCACGAGGCCCCCGCCGGACTCCACCAGTTGGACCTCGC

[0770] TGCCCCCGCCCCCGGACCCGCCCCCGCCGCTCCCGCCCCCGCCCTTAATC

[0771] TCGAGTTTGGTCCCGCCCCCAAATGTGTAGGGCAGCTTGCTGTACTGCTGA

[0772] CAGTAGTAGGTGGCGAAGTCCTCAGGCTGCAGGCTGCTGATGGTCAGGGT

[0773] GTAGTCGGTGCCGCTGCCGCTGCCGCTGAATCTGCTAGGCACGCCGCTGT

[0774] GCAGGCTGCTTGTGTAGTAGATCAGCAGCTTCACGGTCTTGTCGGGCTTCT

[0775] GCTGATACCAGTTCAGGTAGTTGCTGATGCCTTGGGAGGCGCTGCAGGTG

[0776] ATGGTCACTCTGTCGCCCACGGAGGCGCTCAGGCTGCTAGGGCTCTGTGT

[0777] CATCTGAATGTCCGGCCTGGCGGCGTGGAGCAGCAAGGCCAGCGGCAGGA

[0778] GCAAGGCGGTCACTGGTAAGGCCATGGTGGCAGCGCTCTAGAACCGGTCC

[0779] TGTGTTCTGGCGGCAAACCCGTTGCGAAAAAGAACGTTCACGGCGACTAC

[0780] TGCACTTATATACGGTTCTCCCCCACCCTCGGGAAAAAGGCGGAGCCAGTA

[0781] CACGACATCACTTTCCCAGTTTACCCCGCGCCACCTTCTCTAGGCACCCGT

[0782] TCAATTGCCGACCCCTCCCCCCAACTTCTCGGGGACTGTGGGCGATGTGC

[0783] GCTCTGCCCACGGACAGACGGTTCCGGGGCCGCATCGACTTCTCAGGGTC

[0784] CCAGGACAACCTGACTATCACCATGCACCGCCTGCAGCTGTCGGACACTG

[0785] GCACCTACACCTGCCAGGCCATCACGGAGGTCAATGTCTACGGCTCCGGC

[0786] ACCCTGGTCCTGGTGACAGGTAGGGAATGTGCCCATCCCAGACCCCCCTC

[0787] CCAACCCCAGCTGCTGGCCAGGCTCTGCTCCCCCAGCCCTTGTCGTGGGA

[0788] CCCTCCCTCCTACATGTGCCTGAACTGTTCCAGCTCCCAGCCCACTGCCCC

[0789] CAGCAGCCTCCTAGATAGCTGCCCCTCCTCCCCTCCACAGCCTTTCCCTGC

[0790] CCCGAATCCCAAACCCCGGGGGCTCTAACAGGTTCTCCACCGGGAGAATC

[0791] CCTTCCTTCTTTTTTCCTTCTCAGAGGAACAGTCCCAAGGATGGCACAGAT

[0792] GCTCGGACGCCCCACCAAGGGCCTCTGCCCTCCCTGCCCCACCGACAGGC

[0793] TCCGCCCTCCCTGACCCGCAGACAGCCTCTGCCCTCCCTGACCCGCCAGCA

[0794] GCCTCTGCCCTCCCTGCGGCCCTGGCGGTGATCTCCTTCCTCCTCGGGCTG

[0795] GGCCTGGGGGTGGCGTGTGTGCTGGCGAGGACACAGGTCAGTGTGAGCCC

[0796] CAGCTGCCACCTGCACCCCAAAGATTGTTCCCTCTCCTGAGAGCAGCGTGG

[0797] GGGGCGCCAAAGCCCCAGAGGAAAACCCTGGTACCCGCCGCCTCCTCTGC

[0798] AGGCTCTGGAACTCTCAGGGTGGAAGTGGCCTCGGCGGGGAGGGAGGGG

[0799] GACC

[0800] SEQ ID NO:34 - Candidate enhanced CAR structure 6 - group template dsDNA (CD28 - CD7 - CD3ζ);

[0801] CACCGCCTCCAGGAAGCCCTCTCTGAGCTCTGAGCGCCTGCGGTCTCCTGT

[0802] GTGCTGCTCTCTGTGGGGTCCTGTAGACCCAGAGAGGCTCAGCTGCACTC

[0803] GCCCGGCTGGGAGAGCTGGGTGTGGGGAACATGGCCGGGCCTCCGAGGC

[0804] TCCTGCTGCTGCCCCTGCTTCTGGCGCTGGCTCGCGGCCTGCCTGGGGCC

[0805] CTGGCTGCCCAAGGTAAGAGCTTCCCAGGCTCTCCATGGCCACAGCTCCA

[0806] GAGCTCTCCCTGCCCCATGAGCTCAGAGCCCCCAGTCTGAGCCACAGCAC

[0807] AGCCCCCAGGAAGCGGGTGGGGTGCTGAGCGGCCTCCAGTGTCTGAGGAC

[0808] TCATTTAAGAGAAGGAAAAAGGGTGGACCCCGGTGGGGAGTGGCCGGGGC

[0809] TGTCCAGGCAGGGCCGCTGCTTTGGGAGGAAGAAGCCCACAGTCTCGGAA

[0810] CACGAGGACAGCACCTCCCCCAACACCACAGCCGGTGCCCAGATCTGCTC

[0811] CATGCCCCGTAAGGCACCGTGTCTTTGGCGACATGTCAGCCCTGGGCTGT

[0812] CTCAGGGCCCCACCATCCCCACCACTGTCCCCTGCAGGGAGGACATTCTCT

[0813] GTCCTTCTGGCCAGACTGATGGTGACAGCCCAGGTCCTCCCCAGAGGTGC

[0814] AGCAGTCTCCCCACTGCACGACTGTCCCCGTGGGAGCCTCCGTCAACATCA

[0815] CCTGCTCCACCAGCGGGGGCCTGCGTGGGATCTACCTGAGGCAGCTCGGG

[0816] CCACAGCCCCAAGACATCATTTACTACGAGGACGGGGTGGTGCCCACCCA

[0817] TAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCC

[0818] CCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCTACTCA

[0819] GACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGG

[0820] CACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGC

[0821] TGGCAACTAGAAGGCACAGTTAGCGAGGGGGCAGGGCCTGCATGTGAAGG

[0822] GCGTCGTAGGTGTCCTTGGTGGCTGTACTGAGACCCTGGTAAAGGCCATC

[0823] GTGCCCCTTGCCCCTCCGGCGCTCGCCTTTCATCCCAATCTCACTGTAGGC

[0824] CTCCGCCATCTTATCTTTCTGCAGTTCATTGTACAGGCCTTCCTGAGGGTT

[0825] CTTCCTTCTCGGCTTTCCCCCCATCTCAGGGTCCCGGCCACGTCTCTTGTC

[0826] CAAAACATCGTACTCCTCTCTTCGTCCTAGATTGAGCTCGTTATAGAGCTG

[0827] GTTCTGGCCCTGCTGGTACGCGGGGGCGTCTGCGCTCCTGCTGAACTTCA

[0828] CTCTCTGGTACTGGTTGGGGGAGGACAGCGTGTTGCAGCGGCTGTGCGAC

[0829] ATGTCCTCGTACACCACACATGCCGCCGAATTCTTATCCCGCCACGAGCAC

[0830] AGTTTCTTTATCTGTGTCCTGGAGCGATAGGCTGCGAAGTCGCGTGGTGG

[0831] GGCATAGGGCTGGTAATGCTTGCGGGTGGGCCCGGGGCGGCGGGGAGTC

[0832] ATGTTCATGTAGTCACTGTGCAGGAGCCTGCTCCTCTTACTCCTGCAGTAA

[0833] AGGGTGATAACCAGTGACAGGAGAAGGACCCCACAAGTCCCGGCCAAGGG

[0834] CGCCCAGATGTAGATATCACAGGCGAAGTCCAGCCCCCTCGTGTGCACTG

[0835] CGCCCCCCGCCGCTGGCCGGCACGCCTCTGGGCGCAGGGACAGGGGCTGC

[0836] GACGCGATGGTGGGCGCCGGTGTTGGTGGTCGCGGCGCTGGCGTCGTGGT

[0837] GCTGCTCACGGTCACGGTGGTGCCTTGGCCCCACACGTCCAGGTAGCCTC

[0838] TCACCTCGTCTCTAGCGCAGTAGTACACGGCGGTGTCCTCGGCTCTCAGGC

[0839] TGTTCATCTGCAGGTACAGGCTGTTCTTGGCGTTGTCTCTGCTGATGGTGA

[0840] ATCTGCCCTTCACGCTGTCGGGGTAGTAGGTGAAGCCGCCGCTGCTGATG

[0841] CTGCTCACCCACTCCAGTCTCTTCTCGGGGGCTTGTCTCACCCAGCTCATG

[0842] GCGTAGCTGCTGAAGGTCAGGCCGGAGGCGGCGCAGCTCAGTCTCAGGCT

[0843] GCCGCCAGGCTTCACGAGGCCCCCGCCGGACTCCACCAGTTGGACCTCGC

[0844] TGCCCCCGCCCCCGGACCCGCCCCCGCCGCTCCCGCCCCCGCCCTTAATC

[0845] TCGAGTTTGGTCCCGCCCCCAAATGTGTAGGGCAGCTTGCTGTACTGCTGA

[0846] CAGTAGTAGGTGGCGAAGTCCTCAGGCTGCAGGCTGCTGATGGTCAGGGT

[0847] GTAGTCGGTGCCGCTGCCGCTGCCGCTGAATCTGCTAGGCACGCCGCTGT

[0848] GCAGGCTGCTTGTGTAGTAGATCAGCAGCTTCACGGTCTTGTCGGGCTTCT

[0849] GCTGATACCAGTTCAGGTAGTTGCTGATGCCTTGGGAGGCGCTGCAGGTG

[0850] ATGGTCACTCTGTCGCCCACGGAGGCGCTCAGGCTGCTAGGGCTCTGTGT

[0851] CATCTGAATGTCCGGCCTGGCGGCGTGGAGCAGCAAGGCCAGCGGCAGGA

[0852] GCAAGGCGGTCACTGGTAAGGCCATGGTGGCAGCGCTCTAGAACCGGTCC

[0853] TGTGTTCTGGCGGCAAACCCGTTGCGAAAAAGAACGTTCACGGCGACTAC

[0854] TGCACTTATATACGGTTCTCCCCCACCCTCGGGAAAAAGGCGGAGCCAGTA

[0855] CACGACATCACTTTCCCAGTTTACCCCGCGCCACCTTCTCTAGGCACCCGT

[0856] TCAATTGCCGACCCCTCCCCCCAACTTCTCGGGGACTGTGGGCGATGTGC

[0857] GCTCTGCCCACGGACAGACGGTTCCGGGGCCGCATCGACTTCTCAGGGTC

[0858] CCAGGACAACCTGACTATCACCATGCACCGCCTGCAGCTGTCGGACACTG

[0859] GCACCTACACCTGCCAGGCCATCACGGAGGTCAATGTCTACGGCTCCGGC

[0860] ACCCTGGTCCTGGTGACAGGTAGGGAATGTGCCCATCCCAGACCCCCCTC

[0861] CCAACCCCAGCTGCTGGCCAGGCTCTGCTCCCCCAGCCCTTGTCGTGGGA

[0862] CCCTCCCTCCTACATGTGCCTGAACTGTTCCAGCTCCCAGCCCACTGCCCC

[0863] CAGCAGCCTCCTAGATAGCTGCCCCTCCTCCCCTCCACAGCCTTTCCCTGC

[0864] CCCGAATCCCAAACCCCGGGGGCTCTAACAGGTTCTCCACCGGGAGAATC

[0865] CCTTCCTTCTTTTTTCCTTCTCAGAGGAACAGTCCCAAGGATGGCACAGAT

[0866] GCTCGGACGCCCCACCAAGGGCCTCTGCCCTCCCTGCCCCACCGACAGGC

[0867] TCCGCCCTCCCTGACCCGCAGACAGCCTCTGCCCTCCCTGACCCGCCAGCA

[0868] GCCTCTGCCCTCCCTGCGGCCCTGGCGGTGATCTCCTTCCTCCTCGGGCTG

[0869] GGCCTGGGGGTGGCGTGTGTGCTGGCGAGGACACAGGTCAGTGTGAGCCC

[0870] CAGCTGCCACCTGCACCCCAAAGATTGTTCCCTCTCCTGAGAGCAGCGTGG

[0871] GGGGCGCCAAAGCCCCAGAGGAAAACCCTGGTACCCGCCGCCTCCTCTGC

[0872] AGGCTCTGGAACTCTCAGGGTGGAAGTGGCCTCGGCGGGGAGGGAGGGG

[0873] GACC。

Claims

1. An enhanced chimeric antigen receptor comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain, wherein the intracellular domain comprises a co-stimulatory signal transduction region and an intracellular signal transduction region, wherein: The intracellular domain also contains an amino acid sequence derived from the CD7 protein.

2. The enhanced chimeric antigen receptor according to claim 1, wherein the amino acid sequence derived from the CD7 protein is an amino acid sequence derived from the intracellular region of the CD7 protein.

3. The enhanced chimeric antigen receptor according to claim 2, wherein the amino acid sequence derived from the intracellular region of the CD7 protein comprises the amino acid sequence shown in SEQ ID NO:16; optionally, the amino acid sequence derived from the intracellular region of the CD7 protein is shown in SEQ ID NO:

16.

4. The enhanced chimeric antigen receptor according to any one of claims 1-3, wherein the amino acid sequence derived from the CD7 protein is linked between the transmembrane region and the co-stimulatory signal transduction region.

5. The enhanced chimeric antigen receptor according to any one of claims 1-4, The co-stimulatory signal transduction region comprises an amino acid sequence derived from 4-1BB or CD28; optionally, the co-stimulatory signal transduction region comprises an amino acid sequence derived from 4-1BB; more preferably, the co-stimulatory signal transduction region is an amino acid sequence derived from 4-1BB; and / or The intracellular signal transduction region comprises one or more amino acids derived from CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signal transduction region comprises an amino acid sequence derived from CD3ζ; further optionally, the intracellular signal transduction region is an amino acid sequence derived from CD3ζ.

6. The enhanced chimeric antigen receptor according to claim 5, wherein the amino acid sequence derived from 4-1BB comprises the amino acid sequence shown in SEQ ID NO:13; optionally, the amino acid sequence derived from 4-1BB is shown in SEQ ID NO:

13.

7. The enhanced chimeric antigen receptor according to claim 5 or 6, wherein the amino acid sequence derived from CD3ζ comprises the amino acid sequence shown in SEQ ID NO:15; optionally, the amino acid sequence derived from CD3ζ is shown in SEQ ID NO:

15.

8. The enhanced chimeric antigen receptor according to any one of claims 1-7, wherein the intracellular domain comprises the amino acid sequence shown in SEQ ID NO:21; optionally, the amino acid sequence of the intracellular domain is shown in SEQ ID NO:

21.

9. The enhanced chimeric antigen receptor according to any one of claims 1-8, wherein the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, CD8α, and DAP10; optionally, the amino acids of the hinge region are derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:11; even more optionally, the amino acid sequence of the hinge region is shown in SEQ ID NO:

11.

10. The enhanced chimeric antigen receptor according to any one of claims 1-9, wherein the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70, DAP10, 2B4, DNAM-1, and HVEM; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:12; even further optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:

12.

11. The enhanced chimeric antigen receptor according to any one of claims 1-10, wherein the N-terminus of the extracellular antigen recognition domain is further connected to a guide peptide; optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO:1; further optionally, the amino acid sequence of the guide peptide is shown in SEQ ID NO:

1.

12. An enhanced chimeric antigen receptor targeting CD7, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain targeting CD7, wherein the intracellular domain comprises a co-stimulatory signal transduction region and an intracellular signal transduction region, wherein: The intracellular domain also contains an amino acid sequence derived from the CD7 protein.

13. The enhanced chimeric antigen receptor targeting CD7 according to claim 11, wherein the amino acid sequence derived from the CD7 protein is an amino acid sequence derived from the intracellular region of the CD7 protein.

14. The enhanced chimeric antigen receptor targeting CD7 according to claim 13, wherein the amino acid sequence derived from the intracellular region of the CD7 protein comprises the amino acid sequence shown in SEQ ID NO:16; optionally, the amino acid sequence derived from the intracellular region of the CD7 protein is shown in SEQ ID NO:

16.

15. The enhanced chimeric antigen receptor targeting CD7 according to any one of claims 12-14, wherein the amino acid sequence derived from the CD7 protein is linked between the transmembrane region and the co-stimulatory signal transduction region.

16. The enhanced chimeric antigen receptor targeting CD7 according to any one of claims 13-15, The co-stimulatory signal transduction region comprises an amino acid sequence derived from 4-1BB or CD28; optionally, the co-stimulatory signal transduction region comprises an amino acid sequence derived from 4-1BB; further optionally, the co-stimulatory signal transduction region is an amino acid sequence derived from 4-1BB; and / or, The intracellular signal transduction region comprises one or more amino acids derived from CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signal transduction region comprises an amino acid sequence derived from CD3ζ; further optionally, the intracellular signal transduction region is an amino acid sequence derived from CD3ζ.

17. The enhanced chimeric antigen receptor targeting CD7 according to claim 16, wherein the amino acid sequence derived from 4-1BB comprises the amino acid sequence shown in SEQ ID NO:13; optionally, the amino acid sequence derived from 4-1BB is shown in SEQ ID NO:

13.

18. The enhanced chimeric antigen receptor targeting CD7 according to claim 16 or 17, wherein the amino acid sequence derived from CD3ζ comprises the amino acid sequence shown in SEQ ID NO:15; optionally, the amino acid sequence derived from CD3ζ is shown in SEQ ID NO:

15.

19. The enhanced chimeric antigen receptor targeting CD7 according to any one of claims 12-18, wherein the intracellular domain comprises the amino acid sequence shown in SEQ ID NO:21; optionally, the amino acid sequence of the intracellular domain is shown in SEQ ID NO:

21.

20. The enhanced chimeric antigen receptor targeting CD7 according to any one of claims 12-19, wherein the extracellular antigen recognition domain targeting CD7 comprises a CD7 antibody heavy chain variable region and a CD7 antibody light chain variable region, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the CD7 antibody heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 of the antibody heavy chain variable region shown in SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 of the CD7 antibody light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 of the antibody light chain variable region shown in SEQ ID NO:4; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 of the CD7 antibody heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 of the antibody heavy chain variable region shown in SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2, and CDR3 of the CD7 antibody light chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 of the antibody heavy chain variable region shown in SEQ ID NO:4; The amino acid sequences of CDR1, CDR2, and CDR3 in the variable region of the antibody light chain shown in NO:

4.

21. The enhanced chimeric antigen receptor targeting CD7 according to claim 20, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the CD7 antibody heavy chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 of the CD7 antibody light chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; optionally, the amino acid sequences of CDR1, CDR2, and CDR3 of the CD7 antibody heavy chain variable region are shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and the amino acid sequences of CDR1, CDR2, and CDR3 of the CD7 antibody light chain variable region are shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.

22. The enhanced chimeric antigen receptor targeting CD7 according to claim 20 or 21, wherein the CD7 antibody heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:3, and the CD7 antibody light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; optionally, the amino acid sequence of the CD7 antibody heavy chain variable region is as shown in SEQ ID NO:3, and the amino acid sequence of the CD7 antibody light chain variable region is as shown in SEQ ID NO:

4.

23. The enhanced chimeric antigen receptor targeting CD7 according to any one of claims 12-22, wherein the extracellular antigen recognition domain targeting CD7 comprises a humanized CD7 scFv antibody.

24. The enhanced chimeric antigen receptor targeting CD7 according to any one of claims 12-23, wherein the extracellular antigen recognition domain targeting CD7 comprises the amino acid sequence shown in SEQ ID NO:2; optionally, the extracellular antigen recognition domain targeting CD7 is the amino acid sequence shown in SEQ ID NO:

2.

25. The enhanced chimeric antigen receptor targeting CD7 according to any one of claims 12-24, wherein the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, CD8α, and DAP10; optionally, the amino acids of the hinge region are derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:11; even more optionally, the amino acid sequence of the hinge region is shown in SEQ ID NO:

11.

26. The enhanced chimeric antigen receptor targeting CD7 according to any one of claims 12-25, wherein the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, Fc70, DAP10, 2B4, DNAM-1, and HVEM; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:12; even further optionally, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:

12.

27. The enhanced chimeric antigen receptor targeting CD7 according to any one of claims 12-26, wherein the N-terminus of the extracellular antigen recognition domain is further connected to a guide peptide; optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO:1; further optionally, the amino acid sequence of the guide peptide is shown in SEQ ID NO:

1.

28. The CD7-targeting enhanced chimeric antigen receptor according to any one of claims 12-27, wherein the amino acid sequence of the enhanced chimeric antigen receptor is as shown in SEQ ID NO:1-SEQ ID NO:2-SEQ ID NO:11-SEQ ID NO:12-SEQ ID NO:21, with the amino acids linked sequentially from the N-terminus to the C-terminus; optionally, the sequential linkage is a direct linkage.

29. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an enhanced chimeric antigen receptor targeting CD7 according to any one of claims 12-28.

30. A carrier comprising the isolated nucleic acid molecule according to claim 29.

31. An engineered immune effector cell comprising an enhanced chimeric antigen receptor targeting CD7 according to any one of claims 12-28, an isolated nucleic acid molecule according to claim 29, or a vector according to claim 30.

32. The engineered immune effector cell according to claim 31, wherein the engineered immune effector cell is selected from one or more of T lymphocytes, natural killer cells, peripheral blood mononuclear cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, NK cells differentiated from induced pluripotent stem cells, and embryonic stem cells.

33. The engineered immune effector cell according to claim 31 or 32, wherein the engineered immune effector cell is a T lymphocyte; optionally, the T lymphocyte is an autologous T lymphocyte or an allogeneic T lymphocyte.

34. The engineered immune effector cells of claim 33, wherein the allogeneic T lymphocytes comprise CD7 / TRAC double-negative cells obtained through gene editing; further, the allogeneic T lymphocytes comprise 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 95%, 95%, 96%, 97%, 98%, or 99% or more of CD7 / TRAC double-negative cells obtained through gene editing.

35. The engineered immune effector cell according to any one of claims 32-34, wherein the T lymphocyte is an αβ T lymphocyte or a γδ T lymphocyte.

36. A pharmaceutical composition comprising engineered immune effector cells according to any one of claims 31-35 and pharmaceutically acceptable excipients.

37. The pharmaceutical composition of claim 36, wherein the pharmaceutically acceptable excipient comprises a protective agent.

38. The pharmaceutical composition of claim 36, wherein the pharmaceutically acceptable excipient comprises cell cryopreservation solution.

39. The pharmaceutical composition according to any one of claims 36-38, wherein the pharmaceutical composition is an intravenous injection.

40. A method for preparing CD7-targeting, engineered immune effector cells, comprising the step of introducing into immune effector cells a nucleotide sequence encoding an enhanced chimeric antigen receptor targeting CD7 as described in any one of claims 12-28.

41. The preparation method according to claim 40, wherein the immune effector cells are selected from one or more of T lymphocytes, natural killer cells, peripheral blood mononuclear cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, NK cells differentiated from induced pluripotent stem cells, and embryonic stem cells.

42. The preparation method according to claim 41, wherein the immune effector cells are T lymphocytes; optionally, the T lymphocytes are autologous T lymphocytes or allogeneic T lymphocytes.

43. The preparation method according to claim 42, wherein the T lymphocytes are αβT lymphocytes or γδT lymphocytes.

44. The preparation method according to any one of claims 40-43, wherein the method of introducing the nucleotide sequence encoding the enhanced chimeric antigen receptor targeting CD7 according to any one of claims 12-28 into immune effector cells is selected from one or more of the following: a viral method or a non-viral method; optionally, the viral method includes using one or more of the following viral vectors: γ-retroviral vector, lentiviral vector, adenovirus-associated viral vector; the non-viral method includes one or more of the following methods: gene transfer using transposons, gene transduction via mRNA, electroporation.

45. The preparation method according to any one of claims 40-44, wherein the nucleotide sequence encoding the enhanced chimeric antigen receptor according to any one of claims 12-28 is introduced into immune effector cells by a method comprising the following steps: Introducing the CRISPR / Cas system and template DNA into immune effector cells; The CRISPR / Cas system includes the Cas protein, sgRNA and / or chRDNA targeting the CD7 gene, and sgRNA and / or chRDNA targeting the Trac gene. The template DNA contains a nucleotide sequence encoding the enhanced chimeric antigen receptor targeting CD7 as described in any one of claims 12-28.

46. ​​The preparation method according to claim 45, wherein the Cas protein includes one or both of Cas9 protein and Cas12 protein.

47. The preparation method according to claim 45 or 46, wherein the sgRNA and / or chRDNA targeting the Trac gene is a chRDNA sequence as shown in SEQ ID NO:

25.

48. The preparation method according to any one of claims 45-47, wherein the sgRNA and / or chRDNA targeting the CD7 gene is an sgRNA sequence as shown in SEQ ID NO:

26.

49. The preparation method according to any one of claims 45-48, wherein the template DNA is dsDNA; optionally, the dsDNA sequence is as shown in SEQ ID NO:

31.

50. The use of an enhanced chimeric antigen receptor targeting CD7 according to any one of claims 12-28, an isolated nucleic acid molecule according to claim 29, a vector according to claim 30, or an engineered immune effector cell according to any one of claims 31-35 in the preparation of a medicament for treating diseases or conditions associated with CD7 expression.

51. The application of claim 50, wherein the disease or condition associated with CD7 expression is CD7. + Hematologic tumor.

52. The application according to claim 51, wherein the CD7 + Hematologic malignancies are selected from one or more of the following: CD7 + Acute T-lymphoblastic leukemia, CD7 + T-cell lymphoma, CD7 + Acute myeloid leukemia.

53. The application according to claim 52, wherein the CD7 + Acute T-lymphoblastic leukemia includes CD7 + Early-stage pre-T lymphoblastic leukemia and other acute T-lymphoblastic leukemias.

54. The application according to claim 52, wherein the CD7 + T-cell lymphoma is selected from one or more of the following: CD7 + T-lymphoblastic lymphoma, CD7+ extranodal NK / T-cell lymphoma, CD7 + Enteropathy-type T-cell lymphoma, CD7 + Primary cutaneous T-cell lymphoma and CD7 + Peripheral T-cell lymphoma.

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