Fully human chimeric antigen receptor (CAR) targeting both CD5 and CD7 and its applications

Abstract

This article provides information on CARs targeting CD5 and / or CD7, host cells expressing CARs targeting CD5 and / or CD7, their preparation methods, and their applications in the treatment of T-cell-related tumors.

Description

[0001] Cross-citation of related applications

[0002] This application claims priority to Chinese Patent Application No. CN202110904143.5, filed on August 6, 2021, entitled "Dual-target fully human chimeric antigen receptor (CAR) and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the biomedical field, specifically to CARs targeting CD5 and CD7 and host cells expressing CARs targeting CD5 and CD7, as well as their preparation methods and applications. Background Technology

[0004] T-cell malignancies include acute T-lymphoblastic leukemia (T-ALL) and T-cell lymphoma (TCL). T-ALL is a blood disorder caused by abnormal proliferation of T lymphocytes, characterized by its aggressiveness and rapid progression. T-cell lymphoma is a type of malignant tumor of T cells that can develop in lymphoid tissues (such as lymph nodes and spleen) or outside of lymphoid tissues (such as the gastrointestinal tract, liver, nasal cavity, and skin), accounting for approximately 10%–15% of non-Hodgkin's lymphomas, with an even higher proportion in my country. T-cell malignancies have high relapse and mortality rates, and currently there are no effective or targeted treatments. Although various multi-drug combination chemotherapy regimens are available, less than 50% of adults and 75% of children with T-ALL survive for more than 5 years. Allogeneic stem cell transplantation is the only treatment option, but it carries certain risks and cytotoxicity. Currently, chimeric antigen receptor T-cell (CAR-T) technology shows great potential in targeted therapy for patients with B-cell leukemia and lymphoma. [1-5] CAR-T therapy can induce remission and even exert long-term effects in patients, significantly reducing the chance of relapse. Although CAR-T therapy has achieved great success in the treatment of B-cell malignancies, its research and application in T-cell malignancies are very limited, with few studies evaluating the therapeutic effect of CAR-T on T-cell malignancies. CAR-T therapy for T-cell malignancies still faces many challenges. Currently, no tumor-specific antigen has been proven to be universally expressed on tumor T cells, and T-cell malignancies often exhibit heterogeneity, which is highly correlated with relapse after treatment.

[0005] CD5 and CD7 are two promising targets for T-cell-targeted therapy of malignant tumors. [6]Many T-cell malignancies express CD7, providing an attractive target for immunotherapy of T-cell cancers. However, normal T cells, including those used to design CAR-T cells, also express CD7. Because most T-NHL and T-ALL cells highly express CD7, and because CD7 is expressed in approximately 24% of AMLs outside of T-cell malignancies, it is considered a marker of leukemia stem cells and is expressed in the vast majority of natural killer (NK) cells and NKT NHLs and leukemias. [7] CD5 is a pan-T cell marker that is widely overexpressed in most T cell malignancies. [8] In normal cells, CD5 expression is limited to thymocytes, peripheral T cells, and a small subset of B lymphocytes, collectively known as B-1 cells. Furthermore, CD5 is a negative regulator of T cell receptor (TCR) signaling and plays a role in protecting against autoimmunity. [9] . Summary of the Invention

[0006] On the one hand, this article provides a chimeric antigen receptor (CAR) whose antigen-binding domain is capable of specifically binding at least CD5 and / or CD7.

[0007] In some implementations, the antigen-binding domain includes:

[0008] 1) A first antibody or its antigen-binding fragment capable of specifically binding to CD5; and / or

[0009] 2) A second antibody or its antigen-binding fragment capable of specifically binding to CD7; and

[0010] 3) Optionally, a third antibody or its antigen-binding fragment that can specifically bind to another antigen different from CD7 and CD5.

[0011] In some embodiments, the first antibody or its antigen-binding fragment and / or the second antibody or its antigen-binding fragment and / or the third antibody or its antigen-binding fragment are single-chain antibodies (scFv) or single-domain antibodies (sdAb).

[0012] In some embodiments, the first antibody or its antigen-binding fragment is tandemly linked to the second antibody or its antigen-binding fragment via a peptide linker; preferably, the peptide linker comprises the amino acid sequence shown in SEQ ID NO: 26.

[0013] In some embodiments, the antigen-binding domain comprises, from the amino terminus to the carboxyl terminus, the first antibody or its antigen-binding fragment, the peptide linker, and the second antibody or its antigen-binding fragment; or the second antibody or its antigen-binding fragment, the peptide linker, and the first antibody or its antigen-binding fragment.

[0014] In some implementations, the first antibody and / or the second antibody are fully human antibodies.

[0015] In some embodiments, the first antibody or its antigen-binding fragment includes heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 31, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 32, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 33.

[0016] In some embodiments, the second antibody or its antigen-binding fragment includes heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 34, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 35, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 36.

[0017] In some embodiments, the first antibody comprises the amino acid sequence shown in SEQ ID NO: 28 or a functional variant thereof.

[0018] In some embodiments, the second antibody comprises the amino acid sequence shown in SEQ ID NO: 30 or a functional variant thereof.

[0019] In some embodiments, the antigen-binding domain includes the amino acid sequence shown in SEQ ID NO: 38 or 40 or a functional variant thereof.

[0020] In some embodiments, the CAR further includes any one or a combination of signal peptides, extracellular hinge regions, transmembrane regions, intracellular co-stimulatory domains, and intracellular signal transduction domains.

[0021] In some embodiments, the signal peptide, the extracellular hinge region, and the transmembrane region are derived from CD8α; the intracellular co-stimulatory domain is derived from the 4-1BB intracellular domain; and the intracellular signal transduction domain is derived from CD3ζ.

[0022] In some embodiments, the signal peptide includes the amino acid sequence shown in SEQ ID NO: 2, the extracellular hinge region includes the amino acid sequence shown in SEQ ID NO: 4, the transmembrane region includes the amino acid sequence shown in SEQ ID NO: 6, the intracellular co-stimulatory domain includes the amino acid sequence shown in SEQ ID NO: 8, and the intracellular signal transduction domain includes the amino acid sequence shown in SEQ ID NO: 10.

[0023] In some embodiments, the CAR also includes a self-cleaving peptide and a truncated form of the EGFR molecule (tEGFR).

[0024] In some embodiments, the self-cleaving peptide is a T2A peptide, preferably comprising the amino acid sequence shown in SEQ ID NO: 12; the tEGFR comprises the amino acid sequence shown in SEQ ID NO: 16.

[0025] In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO: 20 or 22 or a functional variant thereof.

[0026] On the other hand, this article provides nucleic acid molecules that encode the aforementioned CAR or fragments thereof.

[0027] In some embodiments, the above-mentioned nucleic acid molecules include the nucleotide sequences or degenerate variants thereof shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 37 and 39.

[0028] On the other hand, this article provides expression vectors that include the aforementioned nucleic acid molecules.

[0029] On the other hand, this article provides a method for preparing host cells expressing the above-mentioned CAR, which includes introducing the above-mentioned nucleic acid molecule or the above-mentioned expression vector into the host cell.

[0030] In some embodiments, the method further includes gene knockout of the CD5 and / or CD7 genes of the host cell.

[0031] In some embodiments, the gene knockout is performed using CRISPR technology, with the CD5 gene knockout using sgRNA comprising the sequence shown in SEQ ID NO: 45, and the CD7 gene knockout using sgRNA comprising the sequence shown in SEQ ID NO: 46.

[0032] In some embodiments, the host cell is an immune cell, preferably a T cell or an NK cell.

[0033] On the other hand, this article provides host cells that express the aforementioned CAR or include the aforementioned nucleic acid molecules or expression vectors.

[0034] In some implementations, the host cells substantially do not express or are genetically modified to substantially not express CD5 and / or CD7.

[0035] In some embodiments, the host cell is an immune cell, preferably a T cell or an NK cell.

[0036] On the other hand, this article provides host cells that simultaneously express CARs targeting CD5 and CARs targeting CD7.

[0037] In some embodiments, the CD5-targeting CAR is a first antibody or its antigen-binding fragment that can specifically bind to CD5; the CD7-targeting CAR is a second antibody or its antigen-binding fragment that can specifically bind to CD7.

[0038] In some embodiments, the first antibody or its antigen-binding fragment and / or the second antibody or its antigen-binding fragment are scFv or sdAb.

[0039] In some implementations, the first antibody and / or the second antibody are fully human antibodies.

[0040] In some embodiments, the first antibody or its antigen-binding fragment includes HCDR1, HCDR2 and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 31, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 32 and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 33.

[0041] In some embodiments, the second antibody or its antigen-binding fragment includes HCDR1, HCDR2 and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 34, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 35 and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 36.

[0042] In some embodiments, the first antibody comprises the amino acid sequence shown in SEQ ID NO: 28 or a functional variant thereof; and / or the second antibody comprises the amino acid sequence shown in SEQ ID NO: 30 or a functional variant thereof.

[0043] In some embodiments, the CD5-targeting CAR comprises the amino acid sequence shown in SEQ ID NO: 42 or a functional variant thereof; and / or the second antibody comprises the amino acid sequence shown in SEQ ID NO: 44 or a functional variant thereof.

[0044] In some implementations, the host cells substantially do not express or are genetically modified to substantially not express CD5 and / or CD7.

[0045] In some embodiments, the host cell is an immune cell, preferably a T cell or an NK cell.

[0046] On the other hand, this article provides a method for preparing host cells that simultaneously express CARs targeting CD5 and CARs targeting CD7, including:

[0047] 1) Introduce into the host cells a first expression vector expressing the CD5-targeting CAR and a second expression vector expressing the CD7-targeting CAR; or

[0048] 2) Introduce a third expression vector into the host cell expressing the CAR targeting CD5 and the CAR targeting CD7.

[0049] In some embodiments, the first expression vector comprises the nucleotide sequence shown in SEQ ID NO: 27 or 41 or a degenerate variant thereof; the second expression vector comprises the nucleotide sequence shown in SEQ ID NO: 29 or 43 or a degenerate variant thereof; and the third expression vector comprises the nucleotide sequence shown in SEQ ID NO: 23 or a degenerate variant thereof.

[0050] In some embodiments, the method further includes gene knockout of the CD5 and / or CD7 genes of the host cell.

[0051] In some embodiments, the gene knockout is performed using CRISPR technology, with the CD5 gene knockout using sgRNA comprising the sequence shown in SEQ ID NO: 45, and the CD7 gene knockout using sgRNA comprising the sequence shown in SEQ ID NO: 46.

[0052] In some embodiments, the host cell is an immune cell, preferably a T cell or an NK cell.

[0053] On the other hand, this article provides nucleic acid molecules that include the nucleotide sequence shown in SEQ ID NO: 23 or a degenerate variant thereof.

[0054] On the other hand, this article provides the uses of the aforementioned CARs, nucleic acid molecules, expression vectors, and host cells in the preparation of drugs for the treatment of cancer.

[0055] In some implementations, the cancer expresses CD5 and / or CD7 on its cell surface.

[0056] In some implementations, the cancer is T-cell leukemia or T-cell lymphoma.

[0057] On the other hand, this article provides pharmaceutical compositions comprising the aforementioned host cells and pharmaceutically acceptable carriers.

[0058] On the other hand, this article provides a method for treating cancer in a subject, comprising administering the subject a therapeutically effective amount of the aforementioned host cells or the aforementioned pharmaceutical composition.

[0059] In some implementations, the cancer expresses CD5 and / or CD7 on its cell surface.

[0060] In some implementations, the cancer is T-cell leukemia or T-cell lymphoma.

[0061] The CAR or CAR-T cells described in this article can target at least CD5 and CD7 simultaneously, and can be used to treat tumors that express CD5 or CD7, especially tumors that express both simultaneously. Attached Figure Description

[0062] Figure 1 The knockout efficiency of CD5 and CD7 in T cells was shown in Example 1. Figure 1 A) and the CD4 / CD8 ratio of knockout CD5CD7KO T cells to MOCK T cells (A) Figure 1 B) and phenotypic analysis Figure 1 C) Result.

[0063] Figure 2 A schematic diagram of various CAR expression vector constructs.

[0064] Figure 3 This is a schematic diagram of the process for preparing CAR-T cells.

[0065] Figure 4 The expression of EGFRt in CAR-T cells after knockout and transfection in Example 2 is shown. Figure 4 A) and the expression status of CD5 / CD7 antigens ( Figure 4 B).

[0066] Figure 5 The example shown in Example 3 utilizes APC-CD5 and PE-CD7 antibodies (5A) and antibodies utilizing CD5-rFc, CD7-rFc, Tan5-7-rFc, and Tan7-5-rFc. Figure 5 B) Results of detecting CD5 and CD7 expression in tumor cells.

[0067] Figure 6 The results of CD107a degranulation of CAR-T cells by different target cells are shown. In the results for each target cell, from left to right, the results are those co-incubated with CD5, CD7, Tan5-7, Tan7-5, and Dual CAR-T cells, respectively.

[0068] Figure 7 The study demonstrated the killing effect of CAR-T cells on a variety of target cells.

[0069] Figure 8 The proliferation of each CAR-T / T cell was shown after multiple target cell stimulations.

[0070] Figure 9 The study showed the CAR-T / T cells killing effect on a variety of target cells after repeated stimulation with target cells twice.

[0071] Figure 10 The results of animal experiments using various CAR-T / T cells in a CCRF-CEM-ffluc tumor mouse model in Example 4 are shown. Figure 10 A shows the results of in vivo imaging; Figure 10 B shows the survival rate curve; Figure 10 C shows the weight change curve.

[0072] Figure 11 The results of animal experiments on a mouse model of tumors using each CAR-T / T cell in Example 4 are shown. Figure 11 A shows the mixed tumor cell composition; Figure 11 B shows the killing ability of each group of CAR-T cells against CCRF-CEM MIX; Figure 11 C shows the bioluminescence imaging results used to detect tumor growth; Figure 11 D shows the survival curves of the experimental mice; Figure 11 E shows the weight curve of the experimental mice. Detailed Implementation

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0074] Antibodies are generally immunoglobulins secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (peptides, viruses, bacteria, etc.). These foreign substances are correspondingly called antigens. The basic structure of a classic antibody molecule is a tetramer composed of two identical heavy chains and two identical light chains. Based on the conservation of amino acid sequences, the heavy and light chains are divided into a variable region (V) located at the amino terminus and a constant region (C) located at the carboxyl terminus. The interaction between the variable regions of one heavy chain and one light chain forms the antigen-binding site (Fv). Within the variable region, the composition and sequence of amino acid residues in certain areas are more variable than in other areas (backbone regions, FRs), and these are called hypervariable regions (HVRs). Hypervariable regions are actually the key sites for antibody-antigen binding. Because these hypervariable region sequences are complementary to the antigenic determinants, they are also called complementarity-determining regions (CDRs). Both the heavy and light chains have three complementarity-determining regions, designated HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, respectively. In some cases, "antibody" can also refer to antigen-binding fragments with antigen-binding capabilities, such as scFv, Fab, and F(ab')2. Based on their origin or engineering process, "antibody" can be classified as mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, etc. The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and various antibody derivatives (e.g., antibody-drug conjugates).

[0075] A single-chain antibody (scFv) is composed of the variable regions of the heavy and light chains of an antibody linked together by short peptides to form a single peptide chain. Through proper folding, the variable regions from the heavy and light chains interact non-covalently to form the Fv segment, thus allowing scFv to retain its affinity for the antigen relatively well.

[0076] Single-domain antibodies (sdAbs), also known as VHH antibodies, are antibody molecules that possess antigen-binding capabilities and include a heavy chain variable region but lack a light chain. Structurally, a single-domain antibody can also be considered an antigen-binding fragment of an antibody molecule. They were first discovered in camels, and subsequently, researchers screened antibody libraries (such as phage display libraries) to discover more single-domain antibodies with antigen-binding capabilities. Single-domain antibodies offer several advantages over ordinary antibody molecules (e.g., classic tetrameric antibody molecules) or their antigen-binding fragments, including but not limited to: smaller molecular weight, making them easier to reach tissues or sites in the human body that are difficult for ordinary antibody molecules to access, or the ability to access antigenic epitopes in proteins or peptides that are difficult for ordinary antibody molecules to access; and greater stability, being resistant to changes in temperature and pH, as well as the effects of denaturants and proteases.

[0077] "Human antibodies" or "fully human antibodies" refer to antibodies whose variable and constant regions (if any) are derived from human germline immunoglobulin sequences. Fully human antibodies can be obtained in various ways, such as by isolation directly from the blood of immunized humans, by isolation from transgenic animals (e.g., transgenic mice with human germline immunoglobulin genes introduced and mouse germline immunoglobulin genes removed), or by screening in human phage display libraries.

[0078] Chimeric antigen recessive (CAR) receptors, also known as chimeric T-cell receptors or chimeric immune receptors, are engineered membrane protein receptor molecules that confer desired specificity to immune effector cells, such as the ability to bind to cell surface proteins (e.g., tumor antigens). Chimeric antigen receptors typically consist of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The antigen-binding domain is usually a scFv or sdAb sequence responsible for recognizing and binding to a specific antigen. This antigen-binding domain can be monospecific, meaning it binds specifically to only one antigen, or multispecific (e.g., bispecific), meaning it binds specifically to multiple antigens. In some examples provided in this article, the bispecific extracellular antigen-binding domain specifically binds to both CD5 and CD7. This can be achieved by including antibody fragments (e.g., scFv or sdAb) that target or specifically bind to CD5 and antibody fragments (e.g., scFv or sdAb) that target or specifically bind to CD7 within the extracellular antigen-binding domain. Intracellular signaling domains typically include immune receptor tyrosine activation motifs (ITAMs), such as the signal transduction domain derived from the CD3ζ molecule, responsible for activating immune effector cells and producing a killing effect. Additionally, chimeric antigen receptors may include a signal peptide at the N-terminus responsible for the intracellular localization of nascent proteins, and a hinge region between the antigen-binding domain and the transmembrane domain. Intracellular signaling domains may also include co-stimulatory domains derived from molecules such as 4-1BB or CD28. Accordingly, CAR-expressing T cells are abbreviated as CAR-T. CAR-T cells utilize the CAR expressed on their cell surface to recognize target cells, and upon activation by the target cells, produce a non-MHC-restricted killing effect on the target cells. In one example, the general process of treating a subject (such as a cancer patient) with CAR-T cells is as follows: peripheral blood mononuclear cells (PBMCs) are collected from the subject; T cells are isolated and cultured; the CAR-encoding nucleic acid sequence is introduced via lentiviral transduction; CAR+ cells are further cultured and collected; and the CAR+ cells are reinfused into the subject. Those skilled in the art will know that, in some cases, NK cells can be used in place of T cells to perform this process. Therefore, when referring to CAR-T, NK cells expressing CAR may also be included, depending on the circumstances.

[0079] In this article, "Tan5-7 sdAb" refers to a single-domain antibody that specifically binds CD5 and a single-domain antibody that specifically binds CD7, linked by a linker, wherein the single-domain antibody that specifically binds CD5 is located at the amino terminus of the single-domain antibody that specifically binds CD7. "Tan5-7 sdAb" can serve as the antigen-binding domain or a portion thereof of a Tan5-7 CAR. Similarly, "Tan7-5 sdAb" refers to a single-domain antibody that specifically binds CD7 and a single-domain antibody that specifically binds CD5, linked by a linker, wherein the single-domain antibody that specifically binds CD7 is located at the amino terminus of the single-domain antibody that specifically binds CD5. "Tan7-5 sdAb" can serve as the antigen-binding domain or a portion thereof of a Tan7-5 CAR.

[0080] In this article, "Tan5-7 CAR" refers to a CAR (i.e., a tandem bispecific CAR) whose extracellular antigen-binding domains include a single-domain antibody that specifically binds CD5 and a single-domain antibody that specifically binds CD7, wherein the single-domain antibody that specifically binds CD5 is located at the N-terminus of the single-domain antibody that specifically binds CD7. Similarly, "Tan7-5 CAR" refers to a CAR (tandem bispecific CAR) whose extracellular antigen-binding domains include a single-domain antibody that specifically binds CD7 and a single-domain antibody that specifically binds CD5, wherein the single-domain antibody that specifically binds CD7 is located at the N-terminus of the single-domain antibody that specifically binds CD5. Likewise, this article also considers tandem bispecific (or trispecific) CARs constructed from single-domain antibodies (or scFvs) that specifically bind CD5 and / or CD7 and single-domain antibodies (or scFvs) that specifically bind another antigen (such as other tumor-associated antigens or tumor-specific antigens different from CD5 and CD7).

[0081] In this article, "Dual CAR," also known as "parallel CAR" or "bicistronic CAR," refers to two complete CAR molecules, each containing an antigen-binding domain that binds to a different antigen. This article utilizes a self-cleaving peptide to encode a "Dual CAR" using a single nucleic acid molecule. In one example, the Dual CAR specifically binds to CD5 and CD7 separately. In another example, the Dual CAR specifically binds to CD5 and another antigen (such as a tumor-associated antigen or tumor-specific antigen other than CD5 and CD7). In yet another example, the Dual CAR specifically binds to CD7 and another antigen (such as a tumor-associated antigen or tumor-specific antigen other than CD5 and CD7). This article also considers parallel CARs that specifically bind to CD5 and CD7 and another antigen (such as a tumor-associated antigen or tumor-specific antigen other than CD5 and CD7).

[0082] "Targeted" or "specific binding" refers to the fact that, compared to other molecules present in the environment, antibodies or their antigen-binding fragments, as well as CARs or their antigen-binding domains, have a higher binding affinity for a certain molecule (such as tumor cell surface antigens).

[0083] When referring to proteins such as antibodies or their antigen-binding fragments, a "functional variant" means a sequence with altered amino acid sequences compared to the parent protein, achieved through the substitution, deletion, and / or insertion of one or more, such as 1-30, 1-20, or 1-10, such as 1, 2, 3, 4, or 5 amino acids, while substantially retaining the biological properties (or specific biological activities of interest) of the parent protein before the modification. Those skilled in the art know that such modifications can be made to the amino acid sequence of a protein, and subsequently, through screening, protein variants that retain certain biological activities can be obtained. In some embodiments, the functional variant of the protein retains at least 60%, at least 70%, at least 80%, at least 90%, or even 100% of the biological activity of the parent protein before the modification. For antibodies or their antigen-binding fragments, amino acid substitutions, deletions, and / or insertions can occur in heavy chain CDR regions or light chain CDR regions, or heavy chain FR regions or light chain FR regions, or heavy chain constant regions or light chain constant regions, and the resulting functional variant substantially retains the biological properties of the antibody molecule before the modification, such as antigen-binding capacity. Understandably, the variable regions of the heavy chain or light chain, or each CDR region, of the antibody can be altered individually or in combination. In some embodiments, the amino acid changes in one, two, or all three heavy chain or light chain CDRs do not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the amino acid changes can be amino acid substitutions, such as conserved substitutions. In some embodiments, the functional variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher amino acid sequence identity with the parent antibody.

[0084] When referring to nucleic acid molecules, a "degenerate variant" refers to a nucleic acid molecule that encodes a protein with the same amino acid sequence as its parent nucleic acid molecule. Due to the degeneracy of codons—that is, multiple codons encode the same amino acid—such degenerate variants can be readily obtained by those skilled in the art based on the nucleic acid sequences provided herein. In some embodiments, the degenerate variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher nucleotide sequence identity with the parent nucleic acid molecule.

[0085] When referring to amino acid or nucleotide sequences, the term "sequence identity" (also known as "sequence uniformity") refers to the degree of similarity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), typically expressed as a percentage. Generally, sequence alignment is performed and gaps (if any) are introduced before calculating the percentage of similarity between two amino acid or nucleotide sequences. If, at a certain alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered to be identical or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, sequence identity is obtained by dividing the number of matched positions by the total number of positions in the alignment window. In other algorithms, the number of gaps and / or gap length are also taken into account. For the purposes of this invention, the publicly available alignment software BLAST (available at ncbi.nlm.nih.gov) can be used to obtain the optimal sequence alignment and calculate the sequence identity between two amino acid or nucleotide sequences using default settings.

[0086] In some embodiments, the CD5 single-domain antibody provided herein comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or even 100% sequence identity) with the sequence shown in SEQ ID NO: 28. In some embodiments, the CD7 single-domain antibody provided herein comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or even 100% sequence identity) with the sequence shown in SEQ ID NO: 30.

[0087] In some embodiments, the Tan5-7 sdAb provided herein comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or even 100% sequence identity) with the sequence shown in SEQ ID NO: 38. In some embodiments, the Tan5-7 sdAb provided herein comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or even 100% sequence identity) with the sequence shown in SEQ ID NO: 40.

[0088] CD5 is a type I transmembrane glycosylated protein that plays a crucial role in the negative regulation of T-cell receptor signaling and promotes the survival of both normal and malignant lymphocytes. CD5 is one of the characteristic surface markers of malignant T-cell tumors; 80% of T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphomas express CD5. In this article, CD5 can refer to human CD5, whose GenBank accession number is NM_014207.4. The CD5 protein can also include fragments of CD5, such as its extracellular domain and its components.

[0089] CD7 is a cell surface glycoprotein with a molecular weight of approximately 40 kDa. It belongs to the immunoglobulin superfamily and is expressed on the surface of T cells, NK cells, and other cells such as thymocytes and myeloid cells. It plays an important role in T cell interactions and T cell-B cell interactions during early lymphatic development.

[0090] "Host cell," also known as "engineered cell," refers to any cell of any organism that has been modified, transformed, or manipulated by introducing genes, DNA or RNA sequences, proteins or polypeptides, or other molecules. This article focuses on cells that can be used to express the CARs described herein, including mammalian cells, particularly immune cells such as T cells or natural killer (NK) cells.

[0091] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing technology is a newly emerging RNA-guided DNA editing technique that uses Cas nucleases to edit target genes. The CRISPR gene editing system used in this technology includes a Cas nuclease and a single-guide RNA (sgRNA), and may also include ssDNA as a repair template, depending on the situation. A portion of the sgRNA sequence binds to the Cas nuclease, while another portion is complementary to a portion of the target gene sequence. The recognition function of the sgRNA allows the Cas nuclease to form single-stranded or double-stranded cuts at specific sites on the target gene. Cells typically repair broken strands of DNA in two ways: homology-directed repair (HDR) and non-homologous end joining (NHEJ). When using CRISPR technology, for example, to knock out CD5 and CD7 genes in cells, it is usually only necessary to consider disrupting the normal coding function of the CD5 and CD7 genes, such as causing frameshift mutations or gene segment deletions. Typically, after introducing Cas nucleases (such as Cas9) and sgRNA into cells, cells that do not express the product of the gene to be knocked out (such as the CD5 gene) can be selected. Besides CRISPR technology, other techniques can also be used to achieve gene knockout, such as homologous recombination and TALEN technology.

[0092] "Self-cleaving peptides" refer to short peptides that can cleave proteins by ribosome jumping rather than protease hydrolysis, and can include T2A, F2A and P2A, etc.

[0093] The “CSF2RA signal peptide”, or colony stimulating factor 2 receptor alpha subunit alpha signal peptide, can guide the expression of newly synthesized proteins such as EGFRt on the surface of CAR-T cells.

[0094] "EGFRt" or "tEGFR" are used interchangeably in this document to refer to the gene encoding a truncated human epidermal growth factor receptor polypeptide or its encoded product, which lacks the distal membrane EGF-binding domain and cytoplasmic signaling tail but retains the extracellular domain recognized by anti-EGFR antibodies. EGFRt can be used as a non-immunogenic selection tool and a tracking marker for genetically modified cells. In this document, it serves as a marker molecule for CAR-T cells and can also be used to deplete CAR-T cells in vivo when needed, for example, via the ADCC pathway mediated by EGFR antibodies (e.g., cetuximab) (see US8802374B2), i.e., as a safety switch during clinical translation.

[0095] "And / or" should be understood as meaning any one or both of the options.

[0096] "Comprising" or "including" means to include the stated elements, integers, or steps, but does not exclude any other elements, integers, or steps. When "comprising" or "including" is used, it also covers situations consisting of the mentioned elements, integers, or steps, unless otherwise specified. For example, when referring to an antibody variable region that "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0097] "Approximately" usually refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0098] Targeting CD5 and CD7, the targets of T-cell malignancies, we have developed specific single-domain antibodies and prepared CARs using these single-domain antibodies. In some embodiments, the extracellular binding domain of the prepared CAR includes a single-domain antibody targeting CD5. In some embodiments, the extracellular binding domain of the prepared CAR includes a single-domain antibody targeting CD7. In some embodiments, the prepared CAR includes both single-domain antibodies targeting CD5 and single-domain antibodies targeting CD7.

[0099] We also used these CARs and their encoding nucleic acid sequences to prepare CAR-T cells expressing these CARs. In some embodiments, the CAR-T cells express one CAR. In some embodiments, the extracellular binding domain of the CAR expressed by the CAR-T cells includes a single-domain antibody targeting CD5. In some embodiments, the extracellular binding domain of the CAR includes a single-domain antibody targeting CD7. In some embodiments, the extracellular binding domain of the CAR expressed by the CAR-T cells includes both a single-domain antibody targeting CD5 and a single-domain antibody targeting CD7 (e.g., Tan5-7 CAR or Tan7-5 CAR).

[0100] In some embodiments, the CAR-T cell simultaneously expresses two CARs. In some embodiments, the extracellular binding domain of one CAR expressed by the CAR-T cell includes a single-domain antibody targeting CD5, and the extracellular binding domain of the other CAR expressed by the CAR-T cell includes a single-domain antibody targeting CD7 (e.g., Dual CAR).

[0101] Since the T cells used to prepare CAR-T cells may also express CD5 and / or CD7, gene knockout is performed in the T cells used to prepare CAR-T cells to prevent mutual killing. In some embodiments, the CD5 gene of the T cells used to prepare CAR-T cells is knocked out, for example, using CRISPR gene editing technology. In some embodiments, the CD7 gene of the T cells used to prepare CAR-T cells is knocked out. In other embodiments, both the CD5 and CD7 genes of the T cells used to prepare CAR-T cells are knocked out.

[0102] Brief description of amino acid and nucleotide sequences

[0103] The following provides descriptions of the amino acid and encoding nucleic acid sequences of some sdAbs, CARs, and related peptides used in this article (especially in the examples).

[0104] SEQ ID NO: 1 is the nucleic acid sequence encoding the CD8α signal peptide. The CD8α signal peptide can be used to guide newly synthesized CARs to the cell surface. In mature CARs, this signal peptide can be cleaved and therefore may not be included.

[0105] SEQ ID NO: 2 is the amino acid sequence of the CD8α signal peptide.

[0106] SEQ ID NO: 3 is the nucleic acid sequence encoding the CD8α hinge region. The CD8α hinge region can be used to connect the extracellular antigen-binding domain and the transmembrane domain in CARs.

[0107] SEQ ID NO: 4 is the amino acid sequence of the CD8α hinge region.

[0108] SEQ ID NO: 5 is the nucleic acid sequence encoding the transmembrane region of CD8α.

[0109] SEQ ID NO: 6 is the amino acid sequence of the CD8α transmembrane region.

[0110] SEQ ID NO: 7 is the nucleic acid sequence encoding the 4-1BB intracellular domain.

[0111] SEQ ID NO: 8 is the amino acid sequence of the 4-1BB intracellular domain.

[0112] SEQ ID NO: 9 is the nucleic acid sequence encoding the CD3ζ intracellular signal transduction domain.

[0113] SEQ ID NO: 10 is the amino acid sequence of the CD3ζ intracellular signal transduction domain.

[0114] SEQ ID NO: 11 is the nucleic acid sequence encoding the self-cleaving peptide T2A.

[0115] SEQ ID NO: 12 is the amino acid sequence of the self-cleaving peptide T2A.

[0116] SEQ ID NO: 13 is the nucleic acid sequence encoding the CSF2RA signal peptide.

[0117] SEQ ID NO: 14 is the amino acid sequence of the CSF2RA signal peptide.

[0118] SEQ ID NO: 15 is the coding nucleic acid sequence for EGFRt.

[0119] SEQ ID NO: 16 is the amino acid sequence of EGFRt.

[0120] SEQ ID NO: 17 is the nucleic acid sequence encoding the self-cleaving peptide P2A.

[0121] SEQ ID NO: 18 is the amino acid sequence of the self-cleaving peptide P2A.

[0122] SEQ ID NO: 19 is the coding nucleic acid sequence for Tan5-7 CAR.

[0123] SEQ ID NO: 20 is the amino acid sequence of Tan5-7 CAR. Due to the presence of the self-cleaving peptide, the proteins (CAR and EGFRt) on both sides of the self-cleaving peptide separate from each other during translation. This sequence can be considered as the theoretically encoded fusion protein sequence of SEQ ID NO: 19.

[0124] SEQ ID NO: 21 is the coding nucleic acid sequence for Tan7-5 CAR.

[0125] SEQ ID NO: 22 is the amino acid sequence of Tan7-5 CAR. Due to the presence of the self-cleaving peptide, the proteins (CAR and EGFRt) on both sides of the self-cleaving peptide separate from each other during translation. This sequence can be considered as the theoretically encoded fusion protein sequence of SEQ ID NO: 21.

[0126] SEQ ID NO: 23 is the coding nucleic acid sequence for Dual CAR.

[0127] SEQ ID NO: 24 is the amino acid sequence of the Dual CAR. Due to the presence of the self-cleaving peptide, the proteins on both sides of the self-cleaving peptide (the CAR targeting CD5, the CAR targeting CD7, and EGFRt) separate from each other during translation. This sequence can be considered as the theoretically encoded fusion protein sequence of SEQ ID NO: 23.

[0128] SEQ ID NO: 25 is the encoding nucleic acid sequence of the linker. This linker is used to connect a single-domain antibody that specifically binds CD5 and a single-domain antibody that specifically binds CD7 to the antigen-binding domains of Tan5-7 CAR and Tan7-5 CAR.

[0129] SEQ ID NO: 26 is the amino acid sequence of the linker.

[0130] SEQ ID NO: 27 is the encoding nucleic acid sequence of the CD5-61 single-domain antibody (VHH). The CD5-61 single-domain antibody is a specific example of a single-domain antibody targeting CD5 used in the embodiments of this article.

[0131] SEQ ID NO: 28 is the amino acid sequence of CD5-61 VHH.

[0132] SEQ ID NO: 29 is the encoding nucleic acid sequence for CD7-10 VHH. The CD7-10 single-domain antibody is a specific example of a single-domain antibody targeting CD7 used in the embodiments of this article.

[0133] SEQ ID NO: 30 is the amino acid sequence of CD7-10 VHH.

[0134] SEQ ID NO: 31 is the amino acid sequence of CD5-61 HCDR1.

[0135] SEQ ID NO: 32 is the amino acid sequence of CD5-61 HCDR2.

[0136] SEQ ID NO: 33 is the amino acid sequence of CD5-61 HCDR3.

[0137] SEQ ID NO: 34 is the amino acid sequence of CD7-10 HCDR1.

[0138] SEQ ID NO: 35 is the amino acid sequence of CD7-10 HCDR2.

[0139] SEQ ID NO: 36 is the amino acid sequence of CD7-10 HCDR3.

[0140] SEQ ID NO: 37 is the encoding nucleic acid sequence of Tan5-7 sdAb.

[0141] SEQ ID NO: 38 is the amino acid sequence of Tan5-7 sdAb.

[0142] SEQ ID NO: 39 is the encoding nucleic acid sequence of Tan7-5 sdAb.

[0143] SEQ ID NO: 40 is the amino acid sequence of Tan7-5 sdAb.

[0144] SEQ ID NO: 41 is the coding nucleic acid sequence for CD5 CAR.

[0145] SEQ ID NO: 42 is the amino acid sequence of CD5 CAR.

[0146] SEQ ID NO: 43 is the coding nucleic acid sequence for CD7 CAR.

[0147] SEQ ID NO: 44 is the amino acid sequence of CD7 CAR.

[0148] SEQ ID NO: 45 is the nucleotide sequence of sgRNA targeting CD5.

[0149] SEQ ID NO: 46 is the nucleotide sequence of sgRNA targeting CD7.

[0150] Research Overview

[0151] Chimeric antigen receptor T-cell (CAR-T) technology has made groundbreaking progress, with four products—Kymriah, Yescarta, Tecartus, and Breyanzi—approved by the US FDA. However, the scFv in these four CAR-T cell products and the CAR structures currently used in CAR-T clinical trials are mostly derived from other species. Reported CAR-T clinical trial results demonstrate that treatment failure is related to difficulties in CAR-T cell expansion and / or rapid clearance of functional CAR-T cells. The rapid clearance of CAR-T cells, caused by the production of anti-antibodies against scFvs from other species and / or specific T cells in patients after stimulation, may be a major reason for CAR-T treatment failure or early relapse in some patients. To address this issue, we employed fully human single-domain phage display technology to obtain candidate fully human single-domain antibodies for CAR-T cell research. Fully human single-domain CAR-T cells contain only heavy chain antigen recognition domains, have small molecular weights, low risk of immunogenicity, and can be repeatedly infused, potentially prolonging the lifespan of functional CAR-T cells and enabling patients to achieve more durable remission and better prognosis after receiving CAR-T cell therapy.

[0152] Commonly used CARs, in addition to intracellular signal transduction domains (such as CD3ζ), also include one or two co-stimulatory domains, such as those derived from CD28, 4-1BB, ICOS, and / or OX40. The antigen-binding domains of CAR-T cells can transport these domains to the tumor site expressing the targeted antigen, leading to CAR-T cell activation and proliferation, ultimately killing tumor cells expressing the target antigen. This study used CRISPR technology to knock out the CD5 and CD7 genes in T cells and transfected them with tandem / parallel CD5 and CD7 clones. Experimental results showed that CD5+CD7 knockout CAR-T cells could proliferate normally, and CAR-T cells with tandem CD5 and CD7 clones better maintained their killing function against CD5 and CD7 positive target cells. Simultaneously, it minimized CAR-T cell self-activation and suicide phenomena, ensuring its sustainability and effectiveness in clinical validation.

[0153] Example 1. Using CRISPR / Cas9 technology to knock out CD5 and CD7 antigens on the surface of T cells and to separate T cell components. Identification

[0154] (1) Experimental objective and principle:

[0155] CD5 and CD7 are antigens universally expressed on the surface of mature T cells. CD5 CAR-T cells developed by Mamonkin M et al. without CD5 knockout have been reported to exhibit a degree of self-destruction; T cell cannibalization can limit specific CARs, resulting in a limited duration of survival in the patient's body.

[10] However, research by Gomes-Silva et al. indicates that CD7 CAR-T cells without knocking out the CD7 antigen on the T cells themselves cannot be effectively expanded.

[11] To address this issue, this embodiment uses CRISPR / Cas9 technology to simultaneously knock out both CD5 and CD7 genes before CAR expression. This minimizes T cell suicide without affecting CAR-T cell proliferation, and exhibits potent anti-tumor activity and potential for clinical applications. We used CRISPR / Cas9 technology to knock out CD5 and CD7 genes in sorted T cells, and then analyzed the CD4 / CD8 ratio and phenotype of the knockout T cells to determine whether the double knockout affected T cells.

[0156] (2) The brief experimental steps are as follows:

[0157] 1) Select the sgRNA sequence and synthesize primers;

[0158] 2) Synthesize sgRNA by chemical synthesis method.

[0159] 3) sgRNA+Cas9 RNP electroporation of T cells;

[0160] 4) The gene knockout efficiency of sgRNA was detected by FACS after clonal transfection of knockout T cells.

[0161] 5) Detect CD4, CD8, CCR7, and CD45RA in the knockout CD5CD7KO T cells;

[0162] (3) Main materials and reagents:

[0163] EasyEdit sgRNA chemically synthesized by Nanjing Genscript Biotech Co., Ltd.

[0164] CD5 sgRNA:(SEQ ID NO: 45)

[0165]

[0166] CD7 sgRNA:(SEQ ID NO: 46)

[0167]

[0168] TrueCut TM Cas9 Protein v2,thermo,A36498;

[0169] PE anti-human CD8,Biolegend,Cat.No.344706;

[0170] FITC anti-human CD4,Biolegend,Cat.No.980802;

[0171] BV421-anti CCR7,BD,Cat.No.562555;

[0172] APC-CD45RA,Biolegend,Cat.No.304112;

[0173] (4) Experimental results:

[0174] We used Cas9 protein, along with CD5 sgRNA and CD7 sgRNA, to target and knock out CD5 and CD7 in T cells. The knockout efficiency of CD5 and CD7 was assessed using FACS, and we examined whether the knockout affected T cell components. Figure 1 As shown in Figure A, the percentage of cells with double knockout of CD5 and CD7 reached over 80%. To investigate the component differences between double knockout cells and normal MOCK T cells, the expression of CD4 and CD8 in different T cell phenotypes was examined. It was found that there was no significant difference in CD4 and CD8 content between CD5CD7KO T double knockout cells and normal MOCK T cells. Figure 1 B), and there were no significant differences in T cell phenotypes. Figure 1 C).

[0175] Example 2. Lentiviral transfection and EGFR expression detection

[0176] (1) Experimental objective and principle:

[0177] T cells were CD5+CD7 knocked out and transfected with... Figure 2 CAR lentiviral expression vectors with structural features, according to Figure 3 The strategy described involves CD5 and CD7 knockout followed by CAR lentiviral transfection, with efficacy assessed after 5–7 days. The CAR structure contains a CD8α hinge, a CD8α transmembrane region, a 4-1BB co-stimulatory molecule, and CD3ζ, linked to a truncated EGFR molecule (EGFRt) via T2A, which can be used as a safety switch during clinical translation. Because EGFRt is co-expressed with the CAR molecule, it can serve as an indirect indicator of CAR molecule distribution on the T cell surface without affecting the CAR's structure and function. We transfected knockout T cells with CD5, CD7, and tandem clones Tan5-7, Tan7-5, and parallel Dual CAR lentiviral transfection to obtain CAR-carrying CD5CD7KO T cells for subsequent CAR-T function validation.

[0178] (2) The brief experimental steps are as follows:

[0179] 1) Take CAR-T cells cultured to day 5, and 1×10⁻⁶ control CD5CD7KO T and MOCK T cells. 6 Place in a 96-well plate and centrifuge at 600g for 5 minutes.

[0180] 2) Wash twice with PBS, resuspend in 100 μL PBS, add 1 μL each of APC-CD5, PE-CD7 and FITC-EGFR to each well, and incubate at room temperature in the dark for 10 min.

[0181] 3) Wash twice with PBS and then flow cytometry.

[0182] Main materials and reagents:

[0183] APC-CD5 antibody, BD, Cat. No. 555355

[0184] PE anti-human CD7 Antibody,Biolegend,Cat.No.343106

[0185] Alexa Fluor 488anti-human EGFR,Biolegend,Cat.No.352908

[0186] Experimental results:

[0187] Experimental results showed that after lentiviral transfection of CD5CD7KO T double knockout cells, the expression rate of EGFRt exceeded 30%. Figure 4 A). In this study, CAR-T cells target CD5 and CD7. If the CAR-T cells are functioning well, knockout and transfection with lentivirus can eliminate CAR-T / T cells that still express CD5 and CD7 antigens. In this experiment, CD5, CD7, tandem / parallel Tan5-7, Tan7-5, and Dual CAR-T cells can all effectively eliminate other T cells expressing CD5 and CD7 antigens. Figure 4 B).

[0188] Example 3. In vitro functional verification of CAR-T cells

[0189] Target cell selection:

[0190] To assess the function of various CAR-T cells targeting CD5 and CD7, cell lines expressing both CD5 / CD7 and CD5+CD7 are selected for comparison with double-negative cells. Detection is performed using APC-CD5 and PE-CD7 antibodies. Figure 5As shown in Figure A, JURKAT, CCRF, MOLT4, and SUP-T1 were identified as CD5 and CD7 double-positive cells. CCRF-CEM-CD5KO (CCRF-CD5KO) and CCRF-CEM-CD7KO (CCRF-CD7KO) were monoclonal CD5 and CD7 knockout cell lines obtained by CRISPR / Cas9 technology to knock out CD5 and CD7, followed by serial dilution. CCRF-CD5KO was a CD7 single-positive cell, CCRF-CD7KO was a CD5 single-positive cell, and RAJI was a double-negative cell. Antibodies against CD5-rFc, CD7-rFc, Tan5-7-rFc, and Tan7-5-rFc (rFc, Rabbit Fc, antibodies containing a rabbit Fc fragment in their structure) were used to stain various target cells. The results demonstrated that Tan5-7-rFc and Tan7-5-rFc antibodies could bind not only to CD5 / CD7 double-positive target cells but also to CD5 / CD7 single-positive target cells. Figure 5 B).

[0191] Experimental objective and principle:

[0192] Multiple lentiviral vectors targeting CD5 and CD7 proteins, enriched, screened, and identified from a phage antibody library through affinity panning, were constructed into second-generation CARs. Since VHs binding to CD5 and CD7 antigens may not necessarily exhibit good activation function after being constructed into CAR structures, their function in CAR-T cells requires further confirmation and screening to identify the CAR molecules with the best activity. To this end, we prepared lentiviral vectors of these CAR molecule clones and transduced T cells to prepare CAR-T cells. Then, the in vitro biological efficacy of CAR-T cells was evaluated using CD107a degranulation assays and in vitro cytotoxicity assays. Through these functional validations at the CAR-T level, CAR molecules with ideal efficacy and safety were finally screened for downstream CAR-T product development.

[0193] CD107a threshing experiment

[0194] CD107a is a marker of intracellular microvesicles. When microvesicles loaded with granzymes fuse with the cell membrane, the amount of CD107a on the cell membrane increases. When monensin is used to block its reuptake, the intensity of microvesicle release can be quantitatively reflected. When CAR-T cells are stimulated by target antigens on target cells, granzymes are released, and the increase in CD107a can be detected by flow cytometry to determine the activation status of T cells.

[0195] Brief experimental steps for threshing CD107a:

[0196] 1) Centrifuge the CAR-T cells to be tested and the target cells separately at 300g for 5 min at room temperature, discard the supernatant, and resuspend them in 1640 medium + 10% FBS to a concentration of 4 x 10⁻⁶ cells / mL. 6 cells / mL;

[0197] 2) Add 100 μL of the CAR-T cells to be tested and 100 μL of the target cells to a 96-well plate, and mix well.

[0198] 3) Add 1.5 μL of PE / Cy7 mouse anti-human CD107a antibody and 0.2 μL of monensin to each well of cells, and then incubate in a cell culture incubator (37℃, 5% CO2) for 4 h;

[0199] 4) After incubation, centrifuge at 600g for 5 minutes at 4℃, discard the supernatant, and wash the cells twice with 200μL PBS;

[0200] 5) Resuspend the cells in 100 μL PBS, and add 2 μL of APC anti-human EGFR and 2 μL of LBV421 anti-human CD8a antibody respectively. Mix well and incubate on ice in the dark for 20 min.

[0201] 6) After incubation, wash the cells three times with 200 μL PBS; resuspend them in 100 μL PBS and then detect them by flow cytometry.

[0202] Main samples and reagents:

[0203] Target cells: JURKAT, CCRF-CEM, CCRF-CD5 KO, CCRF-CD7 KO, MOLT4, SUP-T1, RAJI;

[0204] Fetal bovine serum (FBS), Gibco, Cat. No. 10099141;

[0205] APC-CD5 antibody, BD, Cat. No. 555355;

[0206] PE anti-human CD7 Antibody,Biolegend,Cat.No.343106;

[0207] Monensin,BioLegend,Cat.No.420701;

[0208] PE / Cy7 mouse anti-human CD107a, BD, Cat.No.561348;

[0209] BV421 anti-human CD8a,BioLegend,Cat.No.301036;

[0210] APC anti-human EGFR, BioLegend, Cat. No. 352906.

[0211] Experimental results:

[0212] CAR-T cells were obtained via lentiviral transduction and cultured in vitro for 9-12 days before CD107a degranulation. The CAR-T cells to be tested, target cells, monensin, and CD107a antibody were co-incubated for 4 hours, with both CAR-T cell and target cell densities at 2 × 10⁶ cells / year. 5 Cells / mL. Samples were then labeled with CD8 and EGFR antibodies before flow cytometry analysis. In Flowjo software, the viable cell gate (P1) was selected from the scatter plot, and cell debris was removed. Cells in the P1 gate were further analyzed to select a single dispersed cell gate (P2). Then, CD8-positive cells were further selected from the P2 gate (P3). Finally, in the P3 gate, the proportion of CD107a-positive cells among EGFR antibody-positive cells (i.e., CAR-positive cells) was analyzed. The analysis results are as follows: Figure 6 As shown, co-incubation of CD5 CAR-T cells with CD5-negative cell lines did not significantly increase degranulation levels, nor did co-incubation of CD7 CAR-T cells with CD7-negative cell lines. However, Tan5-7, Tan7-5, and Dual CAR-T cells could be activated not only by CD5 / CD7 double-positive target cells but also by CD5 / CD7 single-positive target cells. Furthermore, the proportion of CD107a+ cells in Tan5-7 and Tan7-5 CAR-T cells was significantly higher than that in Dual CAR-T cells after stimulation with CD5+CCRF-CD7KO cells.

[0213] In vitro cell killing experiment

[0214] Experimental Objective and Principle: In vitro cell killing assays used CCRF-CEM, JURKAT, MOLT4, and SUP-T1 cells as CD5+CD7+ double-positive target cells, CCRF-CD5 KO as CD7 single-positive target cells, CCRF-CD7KO as CD5 single-positive target cells, and RAJI cells as CD5+CD7 double-negative target cells to evaluate the antigen-specific killing ability of CD5CD7 CAR-T cells. These cells were transduced via lentivirus to obtain target cells stably expressing firefly luciferase; therefore, the luciferase activity in the sample reflects the number of target cells. CAR-T cells and target cells were co-incubated. When target cells are killed by CAR-T cells, luciferase is released and quickly inactivated (firefly luciferase half-life is approximately 0.5 h). If target cells are not killed or inhibited by CAR-T cells, more luciferase will be produced as target cells proliferate and luciferase expression continues. Therefore, the killing effect of CAR-T on target cells can be detected by the activity of luciferase.

[0215] Brief experimental steps for in vitro cell killing:

[0216] 1) Centrifuge the target cells at 300g for 5 min at room temperature, discard the supernatant, and resuspend them in 1640+10% FBS medium to a concentration of 2x10⁻¹⁰. 5 Cells / mL; Add 100 μL of target cells to each well of a 96-well plate;

[0217] 2) Based on the CAR positivity rate and effector-to-target ratio of the CAR-T samples to be tested, add 100 mL of CAR-T cells to each well of a 96-well plate and mix them with the target cells; then incubate in a carbon dioxide incubator for 24 h.

[0218] 3) Use a luciferase assay kit to detect the luciferase activity in each well sample.

[0219] Main samples and reagents:

[0220] Target cells: CCRF-CEM, JURKAT, MOLT4, SUP-T1, CCRF-CD5 KO, CCRF-CD7 KO, and RAJI;

[0221] Steady-Glo Luciferase Assay System, Promega, Cat. No. E2520.

[0222] Experimental results:

[0223] CAR-T cell samples and a fixed number of target cells (2x10) were used. 4CAR-T cells were mixed at different effector-to-target ratios (E:T) and incubated for 24 hours, after which luciferase activity (RLU) in the samples was measured. CD5CD7kT cells served as a control sample containing only target cells. Since luciferase activity reflects the number of target cells in a sample, changes in luciferase activity indicate the CAR-T cells' ability to kill / inhibit target cells. A lower RLU reading indicates more target cells have been killed.

[0224] like Figure 7 As shown, all CAR-T cell samples effectively killed CD5+CD7+ double-positive target cells, and showed no significant killing effect when co-incubated with negative target cells. Among them, Tan5-7, Tan7-5, and Dual CAR-T cells showed stronger killing ability against CD5+CD7+ double-positive target cells than CD5 CAR-T cells and CD7 CAR-T cells, and Tan5-7, Tan7-5, and Dual CAR-T cells could also kill CD5+CD7- single-positive target cells and CD5-CD7+ single-positive target cells.

[0225] Repeated stimulation proliferation experiment

[0226] Experimental objective and principle:

[0227] Mitomycin-treated target cells (CCRF-CEM) were mixed with CD5CD7KOCAR-T cells from different groups, and after multiple stimulations, the CAR-T cells and target cells were co-incubated to determine the proliferative capacity of different CAR-T cells after repeated stimulation by target cells.

[0228] Brief experimental steps:

[0229] 1) Take 7×10⁶ CCRF-CEM cells 6 Cells, 300g, room temperature, centrifuged for 5 minutes;

[0230] 2) Adjust the density of the complete culture medium to 0.2 × 10⁻⁶. 6 Add 5 μL of Mitomycin stock solution (1 μg / μL) to cells / mL, mix well, and culture for 24 h before use.

[0231] 3) After 24 hours of treatment, CCRF-CEM-Mitomycin cells were centrifuged at 300g, the medium was changed, and the cells were washed 6 times with PBS. The CCRF-CEM-Mitomycin cells were then resuspended in CTS medium, counted, and the density was adjusted to 6 × 10⁻⁶. 6 Cells / mL, ready for use.

[0232] 4) Take 3×10 5CAR-T cells were transferred into 24-well plates. 50 μL of CCRF-CEM-Mitomycin cells were added to each well of the CAR-T plate to achieve an effector-to-target ratio (E:T) of 1:1. The culture volume was increased to 500 μL with CTS complete medium, mixed thoroughly, and incubated at 37°C with 5% CO2 for 72 h. The cells were then counted. The target cells (CCRF-CEM) were treated again with Mitomycin. The above steps were repeated, and amplification curves were plotted.

[0233] Main samples and reagents:

[0234] Mitomycin C,MCE,Cat.No.HY-113061;

[0235] CCRF-CEM of target cells;

[0236] Experimental results:

[0237] like Figure 8 As shown, all six groups of CAR-T / T cell samples expanded effectively after repeated stimulation. After six stimulations with target cells, the expansion capacity of Tan5-7 CAR-T cells was stronger than that of Dual CAR-T cells. The proliferative capacity of CAR-T cells after stimulation by target cells is closely related to the long-term prognosis of patients. Therefore, tandem clones of Tan5-7 CAR-T cells can be considered to have a stronger potential for long-term proliferation and tumor cell elimination.

[0238] In vitro killing experiment after repeated stimulation :

[0239] Experimental Objective and Principle: CAR-T / T cells were repeatedly stimulated twice with CCRF-CEM target cells. The proliferated CAR-T / T cells were then used to investigate their cytotoxic effects on CCRF-CEM, CCRF-CD5 KO, and CCRF-CD7 KO cells. Target cells stably expressing firefly luciferase were obtained through lentiviral transduction. The CAR-T cells and target cells were co-incubated after the two stimulations. The cytotoxic effect of CAR-T cells on target cells was detected by the expression of luciferase.

[0240] Brief experimental procedure for lethality experiment: Same as above

[0241] Main samples and reagents:

[0242] Target cells CCRF-CEM, CCRF-CD5KO, CCRF-CD7KO

[0243] Experimental results:

[0244] The results showed that Tan5-7, Tan7-5, and Dual CAR-T cells still maintained their ability to kill CCRF-CEM, CCRF-CD5KO, and CCRF-CD7KO target cells even after being stimulated twice by target cells. Figure 9 Dual CAR-T cells showed weaker killing ability against CCRF-CD7KO than Tan5-7 and Tan7-5 CAR-T cells after two stimulations with target cells, indicating that Tan5-7 and Tan7-5 CAR-T cells can better maintain their killing ability against CD5+ target cells after stimulation with target antigens than Dual CAR-T cells.

[0245] Example 4. Animal Experiment

[0246] Constructing in vivo animal models using single target cells

[0247] Experimental objective and principle:

[0248] 6-week-old NCG mice were injected with 1*10 via the tail vein. 6 We used CCRF-CEM-ffluc tumor cells to construct a T-ALL mouse tumor model, then injected CAR-T / T cells, and regularly performed fluorescence imaging on the experimental mice to record survival and weight changes, so as to compare the inhibition of tumor cells by each CAR-T experimental group.

[0249] Brief experimental steps:

[0250] 1) Inject 1*10 into each experimental mouse 6 One CCRF-CEM-ffluc cell;

[0251] 2) Perform fluorescence imaging 5 days later to observe whether normal tumor formation occurs;

[0252] 3) After tumor formation, CAR-T cells were injected into mice, with 1*10 CAR+ cells per mouse. 6 Each mouse was tested at 200 μL, with 5 mice in each control group (CD5CD7KO T and PBS).

[0253] 4) Perform fluorescence imaging every 7 days, weigh the samples, and calculate the survival rate.

[0254] Experimental results:

[0255] CCRF-CEM-ffluc tumor cells were injected into mice. After tumor formation, CD5, CD7, Tan5-7, Tan7-5, and Dual CAR-T were infused. CD5CD7KO T and PBS were used as control groups. Fluorescence imaging, weight, and statistical analysis were performed on mice in each group weekly. Results are as follows: Figure 10As shown, on day 10 after CAR-T cell injection, all CAR-T cell groups significantly inhibited tumor cell growth. At day 21, only one mouse in the CD5 CAR-T group showed tumor recurrence. Figure 10 A), and the survival rate and body weight of the mice remained stable. Figure 10 B Figure 10 C). This indicates that CD5, CD7, Tan5-7, Tan7-5, and Dual CAR-T cells still possess significant cellular activity in vivo and can effectively control tumor cells.

[0256] Constructing in vivo animal models by mixing target cells

[0257] Experimental objective and principle:

[0258] To simulate antigen escape and tumor heterogeneity frequently observed in T-cell malignancies, this experiment used 50% CCRF-CEM-ffluc + 25% CCRF-CD5 KO-ffluc + 25% CCRF-CD7 KO-ffluc tumor cells, mixed them, and injected them into mice to construct a mouse model with the three mixed tumor cell types. CAR-T / T cells were then injected into the mice, and fluorescence imaging was performed regularly to record survival and weight changes, thereby comparing the inhibitory effects of each CAR-T experimental group on tumor cells.

[0259] Brief experimental steps:

[0260] 1) Inject 1*10 into each experimental mouse 6 One CCRF-CEM MIX (50% CCRF-CEM-ffluc + 25% CCRF-CD5 KO-ffluc + 25% CCRF-CD7 KO-ffluc tumor cells);

[0261] 2) Perform fluorescence imaging 5 days later to observe whether normal tumor formation occurs;

[0262] 3) After tumor formation, CAR-T cells were injected into mice, with 1*10 CAR+ cells per mouse. 6 Each mouse was tested in a 200 μL container, with 5 mice in each control group (CD5CD7KO T and PBS).

[0263] 4) Perform fluorescence imaging every 7 days, weigh the samples, and calculate the survival rate.

[0264] Experimental results:

[0265] Experimental CAR-T cells contain both single-target and dual-target structures. To test the in vitro efficacy of dual-target CAR-T, mice were injected with 1*10- 6A mixture of 50% CCRF-CEM-ffluc + 25% CCRF-CD5 KO-ffluc + 25% CCRF-CD7 KO-ffluc tumor cells ( Figure 11 A) The killing ability of CAR-T cells in each group against CCRF-CEM MIX is as follows: Figure 11 As shown in B.

[0266] On day 5, CD5, CD7, Tan5-7, Tan7-5, and Dual CAR-T cells were inoculated, and CD5CD7KO T and PBS were used as control groups. Fluorescence imaging, weight, and statistical analysis were performed on mice in each group weekly. Results are as follows: Figure 11 As shown, tumor growth was monitored by bioluminescence imaging (BLI) 10 days after CAR-T cell injection. Figure 11 C) Tumors in the single-target CD5 and CD7 CAR-T cell groups showed recurrence, while the dual-target Tan5-7, Tan7-5, and Dual CAR-T cell groups maintained good therapeutic efficacy. This indicates that dual-target CAR-T can effectively delay tumor recurrence in T-cell malignancies due to antigen escape and tumor heterogeneity. Tumor recurrence occurred in the Dual CAR-T cell group at day 21, suggesting that the tandem Tan5-7 and Tan7-5 structures have a more durable effect compared to the parallel Dual CAR-T structure. Overall Kaplan-Meier survival curves. Log-rank test was used for comparison of survival curves. Compared with the CD5CD7KO T and PBS treatment groups, the survival rate of mice treated with CAR-T cells was significantly improved (P<0.001). Figure 11 D). The body weight curves showed no statistically significant difference in body weight changes among the groups of mice. Figure 11 E). Downregulation and loss of CD5 and CD7 antigen expression are very common in tumor T cells. Targeting CD5 and CD7 can effectively address the issues of tumor heterogeneity, target escape, and antigen downregulation. Bispecific CAR-T therapy is the future direction of CAR-T therapy. Therefore, this article uses CRISPR / Cas9 to knock out CD5 and CD7 antigens in T cells, and transduces these T cells with CD5 / CD7 bispecific CARs. This can effectively target and kill CD5+ and / or CD7+ malignant T cells without affecting the survival of effector CAR-T cells.

[0267] The following are some of the nucleotide and amino acid sequences mentioned in this article.

[0268] SEQ ID NO:1

[0269] Sequence Type: DNA

[0270] Comment on the CD8α signal peptide nucleic acid sequence

[0271] Sequence:

[0272]

[0273] Size: 63bp

[0274] SEQ ID NO:2

[0275] Sequence Type: PRT

[0276] Comment on CD8α signal peptide protein sequence

[0277] Sequence:MALPVTALLLPLALLLHAARP

[0278] Size: 21aa

[0279] SEQ ID NO:3

[0280] Sequence Type: DNA

[0281] Comment on the CD8α hinge region nucleic acid sequence

[0282] Sequence:

[0283]

[0284] Size: 165bp

[0285] SEQ ID NO:4

[0286] Sequence Type: PRT

[0287] Comment on the CD8α hinge region protein sequence

[0288] Sequence:

[0289]

[0290] Size: 55aa

[0291] SEQ ID NO:5

[0292] Sequence Type: DNA

[0293] Comment on the CD8α transmembrane region nucleic acid sequence

[0294] Sequence:

[0295]

[0296] Size: 84bp

[0297] SEQ ID NO:6

[0298] Sequence Type: PRT

[0299] Comment: CD8α transmembrane region protein sequence

[0300] Sequence:IYIWAPLAGTCGVLLLSLVITLYCNHRN

[0301] Size: 28aa

[0302] SEQ ID NO:7

[0303] Sequence Type: DNA

[0304] Comment: 4-1BB intracellular domain nucleic acid sequence

[0305] Sequence:

[0306]

[0307] Size: 126bp

[0308] SEQ ID NO:8

[0309] Sequence Type: PRT

[0310] Comment: 4-1BB intracellular domain protein sequence

[0311] Sequence:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0312] Size: 42aa

[0313] SEQ ID NO:9

[0314] Sequence Type: DNA

[0315] Comment: CD3z intracellular signaling domain nucleic acid sequence

[0316] Sequence:

[0317]

[0318]

[0319] Size: 336bp

[0320] SEQ ID NO:10

[0321] Sequence Type: PRT

[0322] Comment: CD3z intracellular signaling domain protein sequence

[0323] Sequence:

[0324]

[0325] LPPR Size: 112aa

[0326] SEQ ID NO:11

[0327] Sequence Type: DNA

[0328] Comment: T2A cleavage peptide nucleic acid sequence

[0329] Sequence:

[0330]

[0331] Size: 54bp

[0332] SEQ ID NO:12

[0333] Sequence Type: PRT

[0334] Comment: T2A protein sequence (cleavage peptide)

[0335] Sequence:EGRGSLLTCGDVEENPGP

[0336] Size: 18aa

[0337] SEQ ID NO:13

[0338] Sequence Type: DNA

[0339] Comment: CSF2RA signal nucleic acid sequence

[0340] Sequence:

[0341]

[0342] Size: 66bp

[0343] SEQ ID NO:14

[0344] Sequence Type: PRT

[0345] Comment: CSF2RA signal protein sequence

[0346] Sequence:MLLLVTSLLLCELPHPAFLLIP

[0347] Size: 22aa

[0348] SEQ ID NO:15

[0349] Sequence Type: DNA

[0350] Comment: EGFRt nucleic acid sequence

[0351] Sequence:

[0352]

[0353]

[0354] Size: 1005bp

[0355] SEQ ID NO:16

[0356] Sequence Type: PRT

[0357] Comment: EGFRt protein sequence

[0358] Sequence:

[0359]

[0360] Size: 335aa

[0361] SEQ ID NO:17

[0362] Sequence Type: DNA

[0363] Comment: P2A nucleic acid sequence

[0364] Sequence:gctactaacttcagcctgctgaagcaggctggtgacgtcgaggagaatcctggcccc

[0365] Size: 57bp

[0366] SEQ ID NO:18

[0367] Sequence Type: PRT

[0368] Comment: P2A protein sequence

[0369] Sequence: ATNFSLLKQAGDVEENPGP

[0370] Size: 19aa

[0371] SEQ ID NO:19

[0372] Sequence Type: DNA

[0373] Comment: Tan5-7 CAR nucleic acid sequence

[0374] Sequence:

[0375]

[0376]

[0377] Size: 2649bp

[0378] SEQ ID NO:20

[0379] Sequence Type: PRT

[0380] Comment: Tan5-7 CAR amino acid sequence

[0381] Sequence:

[0382]

[0383]

[0384] Size: 883aa

[0385] SEQ ID NO:21

[0386] Sequence Type: DNA

[0387] Comment: Tan7-5 CAR nucleic acid sequence

[0388] Sequence:

[0389]

[0390]

[0391] Size: 2649bp

[0392] SEQ ID NO:22

[0393] Sequence Type: PRT

[0394] Comment: Tan7-5 CAR amino acid sequence

[0395] Sequence:

[0396]

[0397] Size: 883aa

[0398] SEQ ID NO:23

[0399] Sequence Type: DNA

[0400] Comment: Dual CAR nucleic acid sequence

[0401] Sequence:

[0402]

[0403]

[0404]

[0405] Size: 3444bp

[0406] SEQ ID NO:24

[0407] Sequence Type: PRT

[0408] Comment: Dual CAR amino acid sequence

[0409] Sequence:

[0410]

[0411] Size: 1148aa

[0412] SEQ ID NO:25

[0413] Sequence Type: DNA

[0414] Comment: Linker nucleic acid sequence for Tan5-7 and Tan7-5

[0415] Sequence:GGGGGGGGGGGGCTCTGGGGGGGGTGGCTCAGGTGGCGGTGGCTCT

[0416] Size: 45bp

[0417] SEQ ID NO:26

[0418] Sequence Type: PRT

[0419] Comment: Linker protein sequence connecting Tan5-7 and Tan7-5

[0420] Sequence: GGGGSGGGGSGGGGS

[0421] Size: 15aa

[0422] SEQ ID NO:27

[0423] Sequence Type: DNA

[0424] Comment: CD5-61 VHH nucleic acid sequence

[0425] Sequence:

[0426]

[0427]

[0428] Size: 354bp

[0429] SEQ ID NO:28

[0430] Sequence Type: PRT

[0431] Comment: CD5-61 VHH protein sequence

[0432] Sequence:

[0433]

[0434] Size: 118aa

[0435] SEQ ID NO:29

[0436] Sequence Type: DNA

[0437] Comment: CD7-10 VHH nucleic acid sequence

[0438] Sequence:

[0439]

[0440] Size: 342bp

[0441] SEQ ID NO:30

[0442] Sequence Type: PRT

[0443] Comment: CD7-10 VHH protein sequence

[0444] Sequence:

[0445]

[0446] Size: 114bp

[0447] SEQ ID NO:31

[0448] Sequence Type: PRT

[0449] Comment: Amino acid sequence of CD5-61 HCDR1

[0450] Sequence: GGTFSNYA

[0451] Size: 8aa

[0452] SEQ ID NO:32

[0453] Sequence Type: PRT

[0454] Comment: Amino acid sequence of CD5-61 HCDR2

[0455] Sequence:ISAYNGDT

[0456] Size: 8aa

[0457] SEQ ID NO:33

[0458] Sequence Type: PRT

[0459] Comment: Amino acid sequence of CD5-61 HCDR3

[0460] Sequence:ARYESMSGQDI

[0461] Size: 11aa

[0462] SEQ ID NO:34

[0463] Sequence Type: PRT

[0464] Comment: Amino acid sequence of CD7-10 HCDR1

[0465] Sequence:GFTFTWAW

[0466] Size: 8aa

[0467] SEQ ID NO:35

[0468] Sequence Type: PRT

[0469] Comment: Amino acid sequence of CD7-10 HCDR2

[0470] Sequence:INPSGGSP

[0471] Size: 8aa

[0472] SEQ ID NO:36

[0473] Sequence Type: PRT

[0474] Comment: Amino acid sequence of CD7-10 HCDR3

[0475] Sequence:ARKDKDD Size:7aa

[0476] SEQ ID NO:37

[0477] Sequence Type: DNA

[0478] Comment: Tan5-7 sdAb nucleic acid sequence

[0479] Sequence:

[0480]

[0481] Size: 741bp

[0482] SEQ ID NO:38

[0483] Sequence Type: PRT

[0484] Comment: Tan5-7 sdAb amino acid sequence

[0485] Sequence:

[0486]

[0487]

[0488] Size: 247aa

[0489] SEQ ID NO:39

[0490] Sequence Type: DNA

[0491] Comment: Tan7-5 sdAb nucleic acid sequence

[0492] Sequence:

[0493]

[0494] Size: 741bp

[0495] SEQ ID NO:40

[0496] Sequence Type: PRT

[0497] Comment: Tan7-5 sdAb amino acid sequence

[0498] Sequence:

[0499]

[0500] Size: 247aa

[0501] SEQ ID NO:41

[0502] Sequence Type: DNA

[0503] Comment: CD5 CAR nucleic acid sequence

[0504] Sequence:

[0505]

[0506]

[0507] Size: 2262bp

[0508] SEQ ID NO:42

[0509] Sequence Type: PRT

[0510] Comment: CD5 CAR amino acid sequence

[0511] Sequence:

[0512]

[0513] Size: 754bp

[0514] SEQ ID NO:43

[0515] Sequence Type: DNA

[0516] Comment: CD7 CAR nucleic acid sequence

[0517] Sequence:

[0518]

[0519] Size: 2250bp

[0520] SEQ ID NO:44

[0521] Sequence Type: PRT

[0522] Comment: CD7 CAR amino acid sequence

[0523] Sequence:

[0524]

[0525] Size: 750aa

[0526] SEQ ID NO:45

[0527] Sequence Type: RNA

[0528] Comment: CD5 sgRNA nucleic acid sequence

[0529] Sequence:

[0530]

[0531] Size: 100bp

[0532] SEQ ID NO:46

[0533] Sequence Type: RNA

[0534] Comment: CD7 sgRNA nucleic acid sequence

[0535] Sequence:

[0536]

[0537] Size: 100bp

[0538] References:

[0539] [1] NEELAPU S S, LOCKE F L, BARTLETT N L, et al. Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma[J]. New England Journal of Medicine, 2017, 377(26): 2531-44.

[0540] [2] JACOBSON C A. CD19 Chimeric Antigen Receptor Therapy for Refractory Aggressive B-Cell Lymphoma[J]. Journal of Clinical Oncology, 2018, 37(4): 328-35.

[0541] [3] HIRAYAMA A V, GAUTHIER J, HAY K A, et al. The response to lymphodepletion impacts PFS in aggressive non-Hodgkin lymphoma patients treated with CD19 CAR-T cells[J]. Blood, 2019, blood-2018-11-887067.

[0542] [4] DAVILA M L, RIVIERE I, WANG X, et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia[J]. Science translational medicine, 2014, 6(224): 224ra25.

[0543] [5]GILL S,FREY N V,HEXNER E O,et al.CD19 CAR-T cells combined withibrutinib to induce complete remission in CLL[J].Journal of ClinicalOncology,2017,35(15_suppl):7509-.

[0544] [6]SCHERER L D,BRENNER M K,MAMONKIN M.Chimeric Antigen Receptors forT-Cell Malignancies[J].Frontiers in Oncology,2019,9(126.

[0545] [7]GOMES-SILVA D,ATILLA E,ATILLA P A,et al.CD7 CAR T Cells for theTherapy of Acute Myeloid Leukemia[J].Mol Ther,2019,27(1):272-80.

[0546] [8]CASALI P,BURASTERO S E,NAKAMURA M,et al.Human lymphocytes makingrheumatoid factor and antibody to ssDNA belong to Leu-1+B-cell subset[J].Science,1987,236(4797):77.

[0547] [9]FREITAS C M T,JOHNSON D K,WEBER K S.T Cell Calcium SignalingRegulation by the Co-Receptor CD5[J].Int J Mol Sci,2018,19(5):

[0548]

[10] MAMONKIN M,ROUCE R H,TASHIRO H,et al.A T-cell-directed chimericantigen receptor for the selective treatment of T-cell malignancies[J].Blood,2015,126(8):983-92.

[0549]

[11] GOMES-SILVA D,SRINIVASAN M,SHARMA S,et al.CD7-edited T cellsexpressing a CD7-specific CAR for the therapy of T-cell malignancies[J].Blood,2017,130(3):285-96.

Claims

1. A chimeric antigen receptor (CAR) having an antigen-binding domain capable of specifically binding at least CD5 and CD7, wherein the antigen-binding domain comprises: 1) The first antibody or its antigen-binding fragment that can specifically bind to CD5; and 2) A second antibody or its antigen-binding fragment that can specifically bind to CD7. Wherein the first antibody or its antigen-binding fragment is a single-domain antibody and the second antibody or its antigen-binding fragment is a single-domain antibody. The first antibody or its antigen-binding fragment includes heavy chain complementarity-determining region 1 (HCDR1), HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 31, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 32, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 33; and The second antibody or its antigen-binding fragment includes heavy chain complementarity-determining region 1 (HCDR1), HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 34, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 35 and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

36.

2. The CAR of claim 1, wherein the antigen-binding domain further comprises a third antibody or an antigen-binding fragment thereof capable of specifically binding to another antigen different from CD7 and CD5.

3. The CAR as described in claim 1 or 2, wherein the third antibody or its antigen-binding fragment is a single-chain antibody (scFv) or a single-domain antibody (sdAb).

4. The CAR of claim 1 or 2, wherein the first antibody or its antigen-binding fragment is tandemly linked to the second antibody or its antigen-binding fragment via a peptide linker.

5. The CAR of claim 4, wherein the peptide linker comprises the amino acid sequence shown in SEQ ID NO:

26.

6. The CAR of claim 4, wherein the antigen-binding domain comprises, from the amino terminus to the carboxyl terminus: The first antibody or its antigen-binding fragment, the peptide linker, and the second antibody or its antigen-binding fragment; or The second antibody or its antigen-binding fragment, the peptide linker, and the first antibody or its antigen-binding fragment.

7. The CAR of claim 1 or 2, wherein the first antibody and / or the second antibody are fully human antibodies.

8. The CAR of claim 1 or 2, wherein the amino acid sequence of the first antibody is as shown in SEQ ID NO:

28.

9. The CAR of claim 1 or 2, wherein the amino acid sequence of the second antibody is as shown in SEQ ID NO:

30.

10. The CAR of claim 1 or 2, wherein the amino acid sequence of the antigen-binding domain is as shown in SEQ ID NO: 38 or 40.

11. The CAR of claim 1 or 2, wherein the CAR further comprises a combination of a signal peptide, an extracellular hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signal transduction domain.

12. The CAR of claim 11, wherein the signal peptide, the extracellular hinge region, and the transmembrane region are derived from CD8α; the intracellular co-stimulatory domain is derived from the 4-1BB intracellular domain; and the intracellular signal transduction domain is derived from CD3ζ.

13. The CAR of claim 11, wherein the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 2, the extracellular hinge region comprises the amino acid sequence shown in SEQ ID NO: 4, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO: 6, the intracellular co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 8, and the intracellular signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:

10.

14. The CAR of claim 1 or 2 further comprises a self-cleaving peptide and a truncated form of the EGFR molecule (tEGFR).

15. The CAR of claim 14, wherein the self-cleaving peptide is a T2A peptide; and the tEGFR comprises the amino acid sequence shown in SEQ ID NO:

16.

16. The CAR of claim 15, wherein the self-cleaving peptide comprises the amino acid sequence shown in SEQ ID NO:

12.

17. The CAR of claim 1 or 2, wherein the CAR comprises the amino acid sequence shown in SEQ ID NO: 20 or 22.

18. A nucleic acid molecule encoding a CAR or a fragment thereof as described in any one of claims 1-17.

19. The nucleic acid molecule of claim 18, comprising the nucleotide sequence or a degenerate variant thereof shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 37 and 39.

20. An expression vector comprising the nucleic acid molecule of claim 18 or 19.

21. A method for preparing a host cell expressing the CAR of any one of claims 1-17, comprising introducing the nucleic acid molecule of claim 18 or 19 or the expression vector of claim 20 into the host cell.

22. The method of claim 21, further comprising knocking out the CD5 and / or CD7 genes of the host cell.

23. The method of claim 22, wherein the gene knockout is performed using CRISPR technology, the gene knockout of the CD5 gene uses sgRNA comprising the sequence shown in SEQ ID NO: 45, and the gene knockout of the CD7 gene uses sgRNA comprising the sequence shown in SEQ ID NO:

46.

24. The method of claim 21 or 22, wherein the host cell is an immune cell.

25. The method of claim 24, wherein the immune cell is a T cell or an NK cell.

26. A host cell expressing the CAR of any one of claims 1-17 or comprising the nucleic acid molecule of claim 18 or 19 or the expression vector of claim 20.

27. The host cell of claim 26, wherein the host cell does not express CD5 and / or CD7.

28. The host cell of claim 27, wherein the host cell is genetically modified not to express CD5 and / or CD7.

29. The host cell of claim 26 or 27, wherein the host cell is an immune cell.

30. The host cell of claim 29, wherein the immune cell is a T cell or an NK cell.

31. Host cells simultaneously expressing a CD5-targeting CAR and a CD7-targeting CAR, wherein the CD5-targeting CAR comprises a first antibody or its antigen-binding fragment capable of specifically binding to CD5; and the CD7-targeting CAR comprises a second antibody or its antigen-binding fragment capable of specifically binding to CD7. Wherein the first antibody or its antigen-binding fragment is a single-domain antibody and the second antibody or its antigen-binding fragment is a single-domain antibody. The first antibody or its antigen-binding fragment includes HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 31, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 32, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

33. The second antibody or its antigen-binding fragment includes HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 34, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 35 and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

36.

32. The host cell of claim 31, wherein the first antibody and / or the second antibody are fully human antibodies.

33. The host cell of claim 31, wherein the amino acid sequence of the first antibody is as shown in SEQ ID NO: 28; and / or the amino acid sequence of the second antibody is as shown in SEQ ID NO:

30.

34. The host cell of claim 31, wherein the amino acid sequence of the CD5-targeting CAR is as shown in SEQ ID NO: 42; and / or the amino acid sequence of the CD7-targeting CAR is as shown in SEQ ID NO:

44.

35. The host cell of claim 31, wherein the host cell does not express CD5 and / or CD7.

36. The host cell of claim 35, wherein the host cell is genetically modified not to express CD5 and / or CD7.

37. The host cell of claim 31, wherein the host cell is an immune cell.

38. The host cell of claim 37, wherein the immune cell is a T cell or an NK cell.

39. A method for preparing host cells that simultaneously express a CAR targeting CD5 and a CAR targeting CD7, comprising: 1) Introduce into the host cells a first expression vector expressing the CAR targeting CD5 and a second expression vector expressing the CAR targeting CD7; or 2) Introduce a third expression vector expressing the CAR targeting CD5 and the CAR targeting CD7 into the host cells. The CD5-targeting CAR comprises a first antibody or its antigen-binding fragment capable of specifically binding to CD5; the CD7-targeting CAR comprises a second antibody or its antigen-binding fragment capable of specifically binding to CD7. Wherein the first antibody or its antigen-binding fragment is a single-domain antibody and the second antibody or its antigen-binding fragment is a single-domain antibody. The first antibody or its antigen-binding fragment includes HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 31, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 32, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

33. The second antibody or its antigen-binding fragment includes HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 34, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 35 and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

36.

40. The method of claim 39, wherein the first expression vector comprises the nucleotide sequence shown in SEQ ID NO: 27 or 41 or a degenerate variant thereof; the second expression vector comprises the nucleotide sequence shown in SEQ ID NO: 29 or 43 or a degenerate variant thereof; and the third expression vector comprises the nucleotide sequence shown in SEQ ID NO: 23 or a degenerate variant thereof.

41. The method of claim 39 or 40 further comprises knocking out the CD5 and / or CD7 genes of the host cell.

42. The method of claim 41, wherein the gene knockout is performed using CRISPR technology, the gene knockout of the CD5 gene uses sgRNA comprising the sequence shown in SEQ ID NO: 45, and the gene knockout of the CD7 gene uses sgRNA comprising the sequence shown in SEQ ID NO:

46.

43. The method of claim 39 or 40, wherein the host cell is an immune cell.

44. The method of claim 43, wherein the immune cell is a T cell or an NK cell.

45. Nucleic acid molecules, including the nucleotide sequence shown in SEQ ID NO: 23 or a degenerate variant thereof.

46. ​​Use of the CAR according to any one of claims 1-17, the nucleic acid molecule according to claim 18, 19 or 45, the expression vector according to claim 20, or the host cell according to any one of claims 26-38 in the preparation of a medicament for the treatment of T-lymphoblastic leukemia.

47. A pharmaceutical composition comprising the host cell and pharmaceutically acceptable carrier as described in any one of claims 26-38.

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