Anti-CD70 chimeric antigen receptor and use thereof

Abstract

A dual-epitope chimeric antigen receptor specifically binding to CD70. Specifically, the chimeric antigen receptor comprises an extracellular antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen-binding domain comprises a first antigen-binding domain targeting CD70 and a second antigen-binding domain targeting CD70. The first antigen-binding domain and the second antigen-binding domain target different epitopes of CD70. The first antigen-binding domain and the second antigen-binding domain are nanobodies or antigen-binding fragments thereof. The present invention further relates to a nucleic acid molecule encoding the CAR, an immune cell expressing the CAR, and use of the CAR and the immune cell for the prevention and / or treatment of CD70-related conditions.

Description

Anti-CD70 chimeric antigen receptor and its applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on and claims priority to CN application number 202410494190.0, filed on April 23, 2024, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention relates to the field of biomedicine, and more specifically, to a dual-epitope chimeric antigen receptor (CAR) that specifically binds to different epitopes of CD70. The invention also relates to nucleic acid molecules encoding such CARs, immune cells expressing such CARs, and the use of these CARs and immune cells for the prevention and / or treatment of CD70-related conditions. Technical Background

[0004] In recent years, significant breakthroughs have been achieved in the fields of gene engineering and cell therapy, with CAR-T cell therapy attracting considerable attention as an exciting technology. The core of CAR-T cell therapy is the introduction of CAR (Chimeric Antigen Receptor) proteins onto the surface of T cells through genetic engineering. These CAR proteins contain an external antigen recognition structure, a transmembrane domain, and an intracellular signal transduction domain. Once CAR-T cells bind to specific antigens on the surface of cancer cells, they trigger the activation, proliferation, and killing mechanisms of T cells, ultimately achieving a therapeutic effect.

[0005] The development of CAR (Chimeric Antigen Receptor) technology has gone through several stages. The earliest CAR structures included an external antigen recognition structure, a transmembrane domain, and an intracellular signal transduction domain. With further research, the CAR structure has been gradually optimized, including the addition of co-stimulatory signals and the modulation of CAR affinity, to improve the activity and specificity of CAR-T cells. Specifically, second-generation CAR-T cells (2000s) introduced co-stimulatory molecules, such as CD28 or 4-1BB, to improve the survival, proliferation, and persistence of CAR-T cells. Second-generation CAR-T cells showed better clinical efficacy. Third-generation CAR-T cells (2010s) further improved the combination of co-stimulatory molecules to optimize CAR-T cell function. This generation was designed to further increase anti-tumor effects and reduce toxic side effects. Fourth-generation CAR-T cells combined with nanobodies (recently) introduced more functional modules, such as antibodies and cytokines, to further enhance the function of CAR-T cells. In addition, nanobodies, as the outer domain of the CAR, have also become an innovative design, enhancing the precision and efficacy of CAR-T cells.

[0006] An antigenic epitope is a specific sequence on the surface of molecules in an organism, typically composed of proteins, polysaccharide chains, or lipids. The structure of antigenic epitopes is highly diverse, depending on the biomolecule in which they reside. In proteins, an antigenic epitope is usually a short chain of amino acids, and its secondary structure may include α-helices, β-sheets, etc. For polysaccharide chains, an antigenic epitope may consist of multiple glycosyl groups, forming specific glycosyl sequences or spatial conformations. This diversity allows different types of antigenic epitopes to be recognized by the immune system, thereby triggering corresponding immune responses. Endogenous antigenic epitopes, present in normal cells and tissues, are the basis for the immune system to distinguish between self and non-self. They include cell surface proteins, nucleic acids, and proteins in organelles. Recognition of these antigenic epitopes usually does not trigger an autoimmune response and is the basis for the immune system to maintain self-tolerance. Exogenous antigenic epitopes originate from the external environment, such as microorganisms, viruses, and parasites. The immune system fights infection and invasion by recognizing these antigenic epitopes. Recognition of exogenous antigenic epitopes usually triggers an inflammatory and immune cell response to clear the source of infection.

[0007] The immune system recognizes antigenic epitopes through specific cells and molecules, most importantly T cells and B cells. T cells recognize antigenic epitopes through T cell receptors (TCRs) on their surface, a process that requires helper molecules such as the major histocompatibility complex (MHC). B cells, on the other hand, recognize antigenic epitopes through antibody molecules on their surface; the variable regions of the antibodies are key to binding to the antigenic epitopes.

[0008] These engineered cells, by modifying a patient's T cells, endow them with a highly specific ability to attack cancer cells, thus demonstrating great potential in cancer treatment. The core of CAR-T cells is their CAR structure, within which the external antigen recognition structure determines the CAR-T cell's ability to recognize specific antigens. This structure activates the CAR-T cell's killing mechanism through the specific binding of antigenic epitopes. However, single-antigen CAR-T cell therapy has limitations when facing heterogeneous tumor populations, especially since conventionally designed CAR structures may fail to effectively contact antigens due to variations in antigen structure. Therefore, accurate recognition of antigenic epitopes is crucial for the success of CAR-T cell therapy.

[0009] CD70 is a member of the tumor necrosis factor receptor (TNFR) superfamily and has the ability to regulate the activation, proliferation, and differentiation of T cells and B cells, playing an important role in maintaining the body's immune response. While CD70 is only transiently expressed in activated lymphocytes under physiological conditions, it is abnormally expressed in various cancers, including renal cell carcinoma, lung cancer, hematopoietic tumors, and central nervous system gliomas. It is closely related to tumor development and prognosis, and could serve as a novel biomarker for early cancer diagnosis, a new target for clinical diagnosis and treatment, and a potential prognostic indicator. Summary of the Invention

[0010] Through in-depth research, the inventors have designed a dual-epitope chimeric antigen receptor that targets different CD70 epitopes by using two anti-CD70 nanobodies or their antigen-binding fragments tandemly as extracellular antigen-binding domains. CAR-T cells prepared in this way exhibit good killing activity against CD70-positive tumor cells and show stronger cytotoxicity than single-epitope CAR-T cells. The following invention is thus provided.

[0011] Chimeric antigen receptor

[0012] On one hand, the present invention provides a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain targets different epitopes of CD70. In some embodiments, the CAR comprises the extracellular antigen-binding domain, the spacer domain, the transmembrane domain, and the intracellular signal transduction domain from the N-terminus to the C-terminus.

[0013] 1. Extracellular antigen-binding domain

[0014] In some embodiments, the chimeric antigen receptor is a dual epitope, and the extracellular antigen-binding domain includes a first antigen-binding domain targeting CD70 and a second antigen-binding domain targeting CD70, wherein the first antigen-binding domain and the second antigen-binding domain target different epitopes of CD70.

[0015] The first antigen-binding domain and the second antigen-binding domain are nanobodies or their antigen-binding fragments, wherein,

[0016] (1) The first antigen-binding domain includes CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3, and the second antigen-binding domain includes CDR1 as shown in SEQ ID NO:8, CDR2 as shown in SEQ ID NO:9, and CDR3 as shown in SEQ ID NO:10.

[0017] (2) The first antigen-binding domain includes CDR1 as shown in SEQ ID NO:8, CDR2 as shown in SEQ ID NO:9, and CDR3 as shown in SEQ ID NO:10, and the second antigen-binding domain includes CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3.

[0018] (3) The first antigen-binding domain includes CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3, and the second antigen-binding domain includes CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:6;

[0019] (4) The first antigen-binding domain includes CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:6, and the second antigen-binding domain includes CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3;

[0020] (5) The first antigen-binding domain comprises CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:6, and the second antigen-binding domain comprises CDR1 as shown in SEQ ID NO:8, CDR2 as shown in SEQ ID NO:9, and CDR3 as shown in SEQ ID NO:10; or

[0021] (6) The first antigen-binding domain includes CDR1 as shown in SEQ ID NO:8, CDR2 as shown in SEQ ID NO:9, and CDR3 as shown in SEQ ID NO:10, and the second antigen-binding domain includes CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:6.

[0022] In some implementations, the CDR described in any of the above implementations is defined by the Kabat numbering system.

[0023] In some embodiments, (1) the first antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:4 or a variant thereof, and the second antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:11 or a variant thereof.

[0024] (2) The first antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:11 or a variant thereof, and the second antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:4 or a variant thereof.

[0025] (3) The first antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:4 or a variant thereof, and the second antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:7 or a variant thereof.

[0026] (4) The first antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:7 or a variant thereof, and the second antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:4 or a variant thereof.

[0027] (5) The first antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:7 or a variant thereof, and the second antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:11 or a variant thereof; or

[0028] (6) The first antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:11 or a variant thereof, and the second antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:7 or a variant thereof.

[0029] The variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the original sequence; in some embodiments, the substitutions are conservative substitutions.

[0030] In some embodiments, (1) the first antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:4, and the second antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:11.

[0031] (2) The first antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:11, and the second antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:4.

[0032] (3) The first antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:4, and the second antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:7.

[0033] (4) The first antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:7, and the second antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:4.

[0034] (5) The first antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:7, and the second antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:11; or

[0035] (6) The first antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:11, and the second antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof as shown in SEQ ID NO:7.

[0036] In some embodiments, the nanobody or its antigen-binding fragment further includes a framework region derived from a camel-derived antibody.

[0037] In other embodiments, the nanobody or its antigen-binding fragment further comprises a heavy chain framework region derived from human immunoglobulins (e.g., a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain germline antibody gene), said heavy chain framework region optionally comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) reversion mutations from human to camel-derived residues. Those skilled in the art will understand that in the nanobody or its antigen-binding fragment V H Sequences whose N-terminus of the H sequence contains or does not contain an amino acid (such as Met) encoded by a start codon are all within the scope of protection of this invention.

[0038] In some implementations, the first antigen-binding domain is directly or via a peptide linker connected to the N-terminus or C-terminus of the second antigen-binding domain.

[0039] In some implementations, the peptide linker is (GmS)n, where m and n are independently integers not less than 0, for example, independently 1, 2, 3 or 4.

[0040] In some embodiments, the peptide linker comprises the sequence shown in SEQ ID NO:29.

[0041] In some embodiments, the extracellular antigen-binding domain comprises the sequence shown in any one of SEQ ID NOs:12-17, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to it, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); in some embodiments, the substitution is a conservative substitution.

[0042] 2. Transmembrane domain

[0043] The transmembrane domain of the CAR of the present invention can be any protein structure known in the art, provided that it is thermodynamically stable in the cell membrane (particularly the eukaryotic cell membrane). The transmembrane domain of the CAR suitable for use in the present invention can be derived from natural sources. In such embodiments, the transmembrane domain can be derived from any membrane-bound or transmembrane protein. Alternatively, the transmembrane domain can be a synthetic, non-naturally occurring protein segment, such as a protein segment primarily containing hydrophobic residues such as leucine and valine.

[0044] In some embodiments, the transmembrane domain is a transmembrane region selected from the following proteins: CD8α, CD4, CD28, CD137, CD80, CD86, CD152, PD1, and combinations thereof.

[0045] In some embodiments, the transmembrane domain comprises the transmembrane region of CD28.

[0046] In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO:25.

[0047] 3. Spacing zone structural domain

[0048] The chimeric antigen receptor of the present invention may include a spacer domain between the extracellular antigen-binding domain and the transmembrane domain.

[0049] In some embodiments, the spacer domain comprises the CH2 and CH3 regions of an immunoglobulin (e.g., IgG1 or IgG4). In such embodiments, without being bound by any particular theory, it is assumed that CH2 and CH3 extend the antigen-binding domain of the CAR from the cell membrane of the cell expressing the CAR, and more accurately mimic the size and domain structure of the native TCR.

[0050] In some embodiments, the spacer domain comprises a hinge domain. The hinge domain can be an amino acid segment typically found between two domains of a protein, which allows the protein to be flexible and allows movement of one or both domains relative to each other. Therefore, the hinge domain can be any amino acid sequence, as long as it provides this flexibility of the extracellular antigen-binding domain and this mobility relative to the transmembrane domain.

[0051] In some embodiments, the hinge domain is a hinge region or portion thereof of a naturally occurring protein. In some embodiments, the hinge domain comprises a hinge region or portion thereof of CD8α, for example, a fragment of at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids containing a hinge region of CD8α. In some embodiments, the spacer domain comprises the amino acid sequence shown in SEQ ID NO:24.

[0052] 4. Intracellular signal transduction domains

[0053] In some embodiments, the intracellular signal transduction domain includes a co-stimulatory signal transduction domain and / or a primary signal transduction domain.

[0054] In this invention, the co-stimulatory signal transduction domain may be an intracellular signal transduction domain derived from a co-stimulatory molecule. In some embodiments, the co-stimulatory signal transduction domain comprises an intracellular signal transduction domain selected from the following proteins: ligands of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA 1, ICOS, CD2, CD7, LIGHT, NKG2C, B7H3, CD83, and combinations thereof.

[0055] In some embodiments, the co-stimulatory signal transduction domain includes an intracellular signal transduction domain of CD28 and / or an intracellular signal transduction domain of CD137.

[0056] In some embodiments, the co-stimulatory signal transduction domain comprises the sequence shown in SEQ ID NO:27.

[0057] In this invention, the primary signal transduction domain can be any intracellular signal transduction domain containing an immune receptor tyrosine activation motif (ITAM). In some embodiments, the primary signal transduction domain is derived from CD3ζ. In some embodiments, the primary signal transduction domain contains the sequence shown in SEQ ID NO:26.

[0058] In some embodiments, the intracellular signal transduction domain comprises a primary signal transduction domain and at least one co-stimulatory signal transduction domain. The primary signal transduction domain and at least one co-stimulatory signal transduction domain may be connected in series to the carboxyl terminus of the transmembrane domain in any order.

[0059] 5. Signal peptides

[0060] In some embodiments, the CAR of the present invention further comprises a signal peptide at its N-terminus. Typically, a signal peptide is a polypeptide sequence to which a linked sequence is targeted to a desired site in the cell. In some embodiments, the signal peptide can target the linked CAR to the cellular secretory pathway and allow the CAR to further integrate and anchor into a lipid bilayer. Signal peptides that can be used for CARs are known to those skilled in the art. In some embodiments, the signal peptide is derived from CD8α, GM-CSF receptor α, or the IgG1 heavy chain. In some embodiments, the signal peptide comprises the sequence shown in SEQ ID NO:28.

[0061] 6. Full-length CAR

[0062] The present invention provides a chimeric antigen receptor capable of specifically binding to CD70. The chimeric antigen receptor is dual-epitope and comprises, from its N-terminus to its C-terminus, a signal peptide, an extracellular antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signal transduction domain. The extracellular antigen-binding domain binds to different epitopes of CD70.

[0063] In some embodiments, the signal peptide is derived from CD8α and contains the sequence shown in SEQ ID NO:28.

[0064] In some embodiments, the spacer region structural domain includes the hinge region of CD8α, comprising the sequence shown in SEQ ID NO:24.

[0065] In some embodiments, the transmembrane domain comprises a transmembrane region of CD28, containing the sequence shown in SEQ ID NO:25.

[0066] In some embodiments, the intracellular signal transduction domain comprises a co-stimulatory signal transduction domain and a primary signal transduction domain, the co-stimulatory signal transduction domain being connected to the N-terminus of the primary signal transduction domain; the co-stimulatory signal transduction domain comprises an intracellular signal transduction domain of CD28, comprising the sequence shown in SEQ ID NO:27; the primary signal transduction domain is derived from CD3ζ, comprising the sequence shown in SEQ ID NO:26.

[0067] In some embodiments, the chimeric antigen receptor comprises a sequence shown in any one of SEQ ID NOs:18-23, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to it, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to it, and the sequence substantially retains at least one biological activity of the amino acid sequence from which it is derived (e.g., the ability to specifically and reactivity of immune effector cells toward CD70-expressing cells in a non-MHC-restricted manner); in some embodiments, the substitution is a conserved substitution.

[0068] Preparation of chimeric antigen receptors

[0069] Methods for generating chimeric antigen receptors and immune effector cells (e.g., T cells) containing such chimeric antigen receptors are known in the art, and detailed descriptions can be found, for example, Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pp. 1-9; Till et al., 2008, Blood, 112:2261-2271; Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCT patent publications WO2012 / 079000, WO2013 / 126726; and US patent publication 2012 / 0213783, all of which are incorporated herein by reference in their entirety. For example, it may include introducing at least one nucleic acid molecule encoding a CAR into cells and expressing that nucleic acid molecule in the cells. For example, the nucleic acid molecule encoding the CAR of the present invention may be contained in an expression vector (e.g., a lentiviral vector) capable of expression in a host cell, such as a T cell, to manufacture the CAR.

[0070] On the other hand, the present invention provides isolated nucleic acid molecules containing nucleotide sequences encoding the chimeric antigen receptor of the present invention. In some embodiments, the isolated nucleic acid molecules encode the chimeric antigen receptor of the present invention.

[0071] Those skilled in the art will understand that, due to the degeneracy of the genetic code, the nucleotide sequence encoding a chimeric antigen receptor of the present invention can have many different sequences. Therefore, unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence.

[0072] On the other hand, the present invention provides a vector containing the isolated nucleic acid molecules of the present invention. In some embodiments, the vector is a cloning vector or an expression vector.

[0073] In some embodiments, the vector contains a nucleotide sequence encoding the chimeric antigen receptor of the present invention.

[0074] In some implementations, the vector is a viral vector.

[0075] In some implementations, the viral vector is a lentiviral vector.

[0076] Engineered immune cells and their preparation methods

[0077] On the other hand, the present invention provides engineered immune cells comprising the chimeric antigen receptor of the present invention, the isolated nucleic acid molecule of the present invention, or the vector of the present invention.

[0078] In some implementations, the engineered immune cells are human immune cells.

[0079] In some embodiments, the engineered immune cells are selected from T cells, NK cells, macrophages, peripheral blood mononuclear cells, hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

[0080] On the other hand, the present invention provides a method for preparing engineered immune cells expressing the chimeric antigen receptor of the present invention, comprising: (1) providing immune cells; and (2) introducing an isolated nucleic acid molecule of the present invention or a vector of the present invention into the immune cells. The isolated nucleic acid molecule or vector contains a nucleotide sequence encoding the chimeric antigen receptor of the present invention.

[0081] In some implementations, the engineered immune cells are human immune cells.

[0082] In some embodiments, the engineered immune cells are selected from T cells, NK cells, macrophages, peripheral blood mononuclear cells, hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

[0083] In some embodiments, in step (1), the immune cells are pretreated; the pretreatment includes sorting, activation, and / or proliferation of the immune cells. In some embodiments, the pretreatment includes contacting the immune cells with anti-CD3 antibodies and anti-CD28 antibodies to stimulate the immune cells and induce their proliferation, thereby generating pretreated immune cells.

[0084] In some embodiments, in step (2), the nucleic acid molecule or vector is introduced into immune cells via viral infection. In other embodiments, in step (2), the nucleic acid molecule or vector is introduced into immune cells via non-viral vector transfection, such as through transposon vector systems, CRISPR / Cas9 vectors, TALEN methods, ZFN methods, electroporation methods, calcium phosphate transfection, DEAE-glucan-mediated transfection, or microinjection.

[0085] In some implementations, after step (2), the method further includes: amplifying the engineered immune cells obtained in step (2).

[0086] Pharmaceutical Composition

[0087] On the other hand, the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor of the present invention, isolated nucleic acid molecules, a carrier or engineered immune cells; and one or more pharmaceutically acceptable excipients.

[0088] In some embodiments, the pharmaceutical composition may also contain additional antitumor drugs.

[0089] In some embodiments, the chimeric antigen receptor, isolated nucleic acid molecule, carrier, or engineered immune cell of the present invention, along with the additional antitumor drug, can be provided as separate components or as a mixture of components in the pharmaceutical composition. Therefore, the chimeric antigen receptor, isolated nucleic acid molecule, carrier, or engineered immune cell of the present invention, along with the additional antitumor drug, can be administered simultaneously, separately, or sequentially.

[0090] In some embodiments, the one or more pharmaceutically acceptable excipients may comprise a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such a sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0091] The pharmaceutical compositions of the present invention may include a "therapeutic effective amount" or a "preventive effective amount" of the chimeric antigen receptor, isolated nucleic acid molecule, carrier, or engineered immune cell of the present invention. A "preventive effective amount" refers to an amount sufficient to prevent, stop, or delay the onset of disease. A "therapeutic effective amount" refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Therapeutic effective amounts may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc.

[0092] Therapeutic applications

[0093] On the other hand, the present invention provides a method for preventing and / or treating CD70-related diseases in subjects, comprising the steps of administering the chimeric antigen receptor, isolated nucleic acid molecule, carrier, engineered immune cell, or pharmaceutical composition of the present invention to a subject in need. The present invention also relates to the use of said chimeric antigen receptor, isolated nucleic acid molecule, carrier, engineered immune cell, or pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of tumors or autoimmune diseases in subjects.

[0094] In some implementations, the CD70-related disease is a tumor or an autoimmune disease.

[0095] In some implementations, the tumor is a CD70-positive tumor.

[0096] In some implementations, the tumor is a solid tumor or a hematogenous tumor.

[0097] In some embodiments, the solid tumor is selected from renal cell carcinoma, lung cancer, glioma, nasopharyngeal carcinoma, gastric cancer, head and neck cancer, skin cancer, colorectal cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, colorectal cancer, or oral squamous cell carcinoma.

[0098] In some embodiments, the hematogenous tumor is selected from Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, or leukemia.

[0099] In some embodiments, the autoimmune disease is selected from arthritis, rheumatoid arthritis, psoriatic arthritis, lupus, lupus nephritis, systemic lupus erythematosus, psoriasis, vitiligo, alopecia areata, inflammatory bowel disease, ulcerative colitis, Crohn's disease, type I diabetes, multiple sclerosis, autoimmune hepatitis, primary biliary cirrhosis, celiac disease, scleroderma, Graves' disease, Hashimoto's thyroiditis, ankylosing spondylitis, myasthenia gravis, Sjögren's syndrome, IgA nephropathy, IgG4-related disease, vasculitis, ANCA-associated vasculitis, uveitis, pemphigus, bullous pemphigoid, or autoimmune hemolytic anemia.

[0100] In some implementations, the subject is a mammal, such as a human.

[0101] In some embodiments, the chimeric antigen receptor, isolated nucleic acid molecule, carrier, engineered immune cell, or pharmaceutical composition may be used alone or in combination with other antitumor drugs.

[0102] The chimeric antigen receptor, isolated nucleic acid molecule, carrier, engineered immune cell, or pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use.

[0103] A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the necessary dose of the chimeric antigen receptor of the present invention, isolated nucleic acid molecules, carriers, engineered immune cells, or pharmaceutical compositions, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0104] The chimeric antigen receptors, isolated nucleic acid molecules, carriers, engineered immune cells, or pharmaceutical compositions of the present invention can be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intrabladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some embodiments, the chimeric antigen receptors, isolated nucleic acid molecules, carriers, engineered immune cells, or pharmaceutical compositions of the present invention are administered by intravenous injection or bolus.

[0105] Terminology Definition

[0106] 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 operational steps used herein, such as cell culture, molecular biology, biochemistry, nucleic acid chemistry, and immunology, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0107] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0108] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0109] As used herein, the term "CD70" refers to the ligand of CD27 (TNFRSF27), a member of the tumor necrosis factor receptor (TNFR) superfamily, which regulates the activation, proliferation, and differentiation of T cells and B cells, playing a crucial role in maintaining the body's immune response. The sequence of CD70 is well known to those skilled in the art (see, for example, NCBI GENBANK database accession number: NC_000019).

[0110] As used herein, the term "camel-derived antibody" refers to antibodies against an antigen produced by camel-dwelling animals (including camels, alpacas, and llamas) after immunization or antigen invasion. Those skilled in the art know that among the antibodies produced by camel-dwelling animals are "heavy-chain antibodies" (HCAbs) lacking the light chain, which contain only a variable domain of the heavy chain of the HCAb (V... H H) and two conventional CH2 and CH3 regions, and V was cloned and expressed separately. H The H region exhibits excellent structural stability and antigen-binding activity. V H H is the smallest known unit that can bind to a target antigen. It can exist stably on its own in vitro and is also known as a single-domain antibody (sdAb) or nanobody.

[0111] As used herein, the term "nanobody" has the meaning commonly understood by those skilled in the art as an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camel antibody or a shark antibody). Typically, a nanobody consists of four framework regions and three complementarity-determining regions, having a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to omit one or both of those framework regions, as long as they substantially maintain antigen binding and specificity. Nanobodies are also called single-domain antibodies (sdAbs), and the two terms are used interchangeably.

[0112] As used herein, the term "antigen-binding fragment" of a nanobody refers to a polypeptide containing a fragment of a nanobody that retains the ability to specifically bind to the same antigen bound by the nanobody, and / or competes with the nanobody for specific binding to the antigen; it is also referred to as the "antigen-binding moiety." See Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. The antigen-binding fragment of the antibodies of the present invention can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the nanobody of the present invention. In some embodiments, the "antigen-binding fragment" of the nanobody may be truncated at the N-terminus or C-terminus compared to the full-length nanobody to contain only a portion of FR1 and / or FR4, or lack one or both of those backbone regions, as long as it substantially retains antigen binding and specificity.

[0113] Antigen-binding fragments of nanobodies can be obtained from a given nanobody (e.g., the nanobody provided by the present invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of nanobodies can be specifically screened in the same manner as for intact nanobodies.

[0114] In this article, unless the context clearly indicates otherwise, when referring to the term "nanobody," it includes not only the complete nanobody but also the antigen-binding fragment of the nanobody.

[0115] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residue in the variable region of an antibody responsible for antigen binding. The nanobody contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), or the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883). For a given nanobody, those skilled in the art will readily identify the CDR defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0116] As used herein, the term “framework region” or “FR” residues refer to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0117] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the “binding rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). The values ​​of KD, kon, and kdis can be measured using any effective method. In some embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). Alternatively, bioluminescent interferometry or Kinexa can be used to measure the dissociation constant.

[0118] As used herein, the term "chimeric antigen receptor (CAR)" refers to a recombinant polypeptide construct comprising at least one extracellular antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, which combines antibody-based specificity against a target antigen (e.g., CD70) with an intracellular domain activating immune effectors to exhibit specific immune activity against cells expressing that target antigen (e.g., CD70). In this invention, the expression "CAR-expressing immune cell" refers to an immune cell that expresses a CAR and has antigen specificity determined by the targeting domain of that CAR. Methods for manufacturing CARs (e.g., for cancer treatment) are known in the art and can be found, for example, Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCT patent publications WO2012 / 079000 and WO2013 / 059593; and U.S. Patent Publication 2012 / 0213783, all of which are incorporated herein by reference in their entirety. In this document, the expression "anti-CD70 CAR" refers to a CAR containing an extracellular antigen-binding domain capable of specifically binding to CD70; the expression "anti-CD70 CAR-T" refers to immune cells (e.g., PBMCs, T cells) expressing the aforementioned CAR.

[0119] As used herein, the term "extracellular antigen-binding domain" refers to a polypeptide capable of specifically binding to a target antigen or receptor. This domain will be able to interact with cell surface molecules. For example, an extracellular antigen-binding domain can be selected to recognize antigens that serve as cell surface markers associated with a specific disease state. Typically, the extracellular antigen-binding domain is an antibody-derived targeting domain.

[0120] As used herein, the term "epitope" refers to a portion of an antigen that can be recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, the epitope can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of a protein, referred to as a linear epitope or a conformational epitope, respectively. In this invention, the term "dual-epitope chimeric antigen receptor" refers to the extracellular antigen-binding domain of the chimeric antigen receptor of this invention being able to recognize and bind two different epitopes of the target antigen or receptor.

[0121] As used herein, the term "intracellular signal transduction domain" refers to a protein portion that transduces effector signals and guides the cell to perform specific functions. Therefore, intracellular signal transduction domains have the ability to activate at least one normal effector function of CAR-expressing immune cells. For example, effector functions of T cells could be cytolytic activity or helper activities, including cytokine secretion.

[0122] As used herein, the term "primary signal transduction domain" refers to a protein motif capable of regulating primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary signal transduction domains acting in a stimulatory manner typically contain a signal transduction motif known to be an immune receptor tyrosine-based activation motif (ITAM). Non-limiting examples of such primary signal transduction domains include TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, DAP10, CD79a, CD79b, and CD66d.

[0123] As used herein, the term "co-stimulatory signaling domain" refers to the intracellular signaling domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule, other than an antigen receptor or Fc receptor, that provides a second signal required for the efficient activation and function of T lymphocytes upon binding to an antigen. Non-limiting examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS), and DAP10.

[0124] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: 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 that can be used as vectors include, but are not limited to, retrotranscriptoviruses (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 but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0125] As used herein, the term "viral vector" is broadly used to refer to nucleic acid molecules (e.g., transfer plasmids) that typically facilitate the transfer or integration of nucleic acid molecules into the genome of a cell, or viral particles that mediate nucleic acid transfer. In addition to nucleic acids, viral particles typically include various viral components and sometimes host cell components. The term "viral vector" can refer to a virus or viral particle capable of transferring nucleic acids into a cell, or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses.

[0126] As used herein, the term "lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements or portions thereof (including LTRs) primarily derived from lentiviruses. In some embodiments, the terms "lentiviral vector" and "lentiviral expression vector" may be used to refer to lentiviral transfer plasmids and / or infectious lentiviral particles. When elements (e.g., cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc.) are mentioned herein, it should be understood that the sequences of these elements are present in the lentiviral particles of the present invention in RNA form and in the DNA plasmids of the present invention in DNA form.

[0127] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0128] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0129] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0130] As used herein, the term "pharmaceuticalally acceptable excipient" means an excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintainers, absorption delayers, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, and their analogues. Absorption delayers include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffer solutions (such as buffered saline), alcohols and polyols (such as glycerol).

[0131] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., a CD70-related disease) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0132] As used herein, the term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) suffers from a disease related to CD70.

[0133] In this invention, unless otherwise specified, the terms "first" (e.g., first antigen-binding domain) and "second" (e.g., second antigen-binding domain) are primarily for distinguishing reference and do not have a typical sequential meaning.

[0134] Beneficial effects of the invention

[0135] This invention utilizes two anti-CD70 nanobodies or their antigen-binding fragments tandemly as extracellular antigen-binding domains to construct a dual-epitope chimeric antigen receptor targeting different CD70 antigen epitopes. This design not only improves the flexibility of the CAR structure but also enables precise binding to tumor-specific antigens. This helps CAR-T cells to more accurately locate and attack tumor cells, reducing collateral damage to normal cells. The dual-epitope CAR of this invention has good permeability, making it easier to penetrate tumor tissue and access epitope binding sites that conventional single-chain antibodies cannot reach, thus improving the biodistribution and anti-tumor efficacy of CAR-T cells in actual treatment. In vitro experiments have also demonstrated that this dual-epitope CAR design has a stronger in vitro killing effect than CARs that recognize a single antigen epitope. Therefore, the anti-CD70 dual-epitope chimeric antigen receptor of this invention has better potential for the prevention and / or treatment of CD70-positive tumors and has significant clinical value.

[0136] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0137] Figure 1 shows a schematic diagram of CAR structural combinations targeting different antigenic epitopes. It includes CD8 Hinge, CD8 intracellular domain, CD28 intracellular domain, and CD3ζ, with CD70 CAR1 and CD70 CAR3 serving as positive controls.

[0138] Figure 2 shows the CAR positivity rate in T cells transduced using the CD70 dual epitope CAR construct.

[0139] Figure 3 shows the expression level of CD70 in 786-0 cells. The left curve represents the detection results of 786-0NC (negative control), and the right curve represents the detection results of 786-0.

[0140] Figure 4 shows the expression level of CD70 in U251 cells. The left curve represents the detection results of U251NC (negative control), and the right curve represents the detection results of U251.

[0141] Figure 5 shows the results of in vitro cytotoxicity assays of exemplary dual-epitope CAR-T cells against 786-0-Luc cells at different effector-to-target ratios. The horizontal axis represents the ratio of CAR-T cells to 786-0-Luc cells, divided into three groups. In each group, the CAR-T cells, from left to right, are: CAR-T cells expressing CD70 CAR1, CD70 CAR3, Tan-biCAR1-6, and Mock T, respectively.

[0142] Figure 6 shows the change in the positivity rate of CAR-T cells after simulated repeated exposure to tumor cell antigens.

[0143] Figure 7 shows the cell killing effect of an exemplary dual-epitope CAR-T cell after four rounds of antigen stimulation.

[0144] Figure 8 shows the TIM-3 positivity rate of exemplary dual-epitope CAR-T cells after four rounds of antigen stimulation. The horizontal axis in the figure represents different CAR-T cell groups, which are divided into 6 groups. In each group, the left side represents before antigen stimulation, and the right side represents after antigen stimulation.

[0145] Figure 9 shows the PD-1 positivity rate of exemplary dual-epitope CAR-T cells after four rounds of antigen stimulation. The horizontal axis in the figure represents different CAR-T cell groups, which are divided into 6 groups. In each group, the left side represents before antigen stimulation, and the right side represents after antigen stimulation.

[0146] Figure 10 shows the results of an in vitro IFN-γ release assay of T cells containing an exemplary dual-epitope CAR targeting 786-0-Luc cells.

[0147] Figure 11 shows the results of an in vitro IL-2 release assay of T cells containing an exemplary dual-epitope CAR targeting 786-0-Luc cells.

[0148] Figure 12 shows the results of an in vitro TNF-α release assay of T cells containing an exemplary dual-epitope CAR targeting 786-0-Luc cells.

[0149] Figure 13 shows the results of in vitro cytotoxicity assays of T cells containing exemplary single-epitope and dual-epitope CARs targeting U251-Luc cells. The horizontal axis in the figure represents the ratio of CAR-T cells to U251-Luc cells, divided into 4 groups. In each group, the CAR-T cells from left to right are: CAR-T cells expressing Tan-biCAR3, Tan-biCAR4, CD70CAR1, CD70 CAR3, and Mock T, respectively.

[0150] Figure 14 shows the percentage of lysis of U251-Luc cells by exemplary dual-epitope CAR-T cells after repeated exposure to tumor cell antigens. The horizontal axis in the figure represents the ratio of CAR-T cells to U251-Luc cells, divided into 4 groups. In each group, the CAR-T cells from left to right are: CAR-T cells expressing Tan-biCAR3, Tan-biCAR4, CD70 CAR1, CD70 CAR3, and Mock T, respectively.

[0151] Figure 15 shows that the proliferation of dual-epitope CAR-T cells containing Tan-biCAR4 after repeated exposure to tumor antigens was significantly higher than that of single-epitope CAR-T cells.

[0152] Figure 16 shows that, upon re-stimulation by the antigen, exemplary dual-epitope CAR-T cells can continue to generate a large number of cloned effector memory T cells (Tem) carrying the same antigen. The test results are grouped according to different CAR-T cell types, with the cell subtypes in each group from top to bottom being Teff, Tem, Tcm, and Tnaive. Mock T serves as the negative control.

[0153] Figure 17 shows that compared to the more lethal single-episode CAR, the dual-episode CAR has lower exhaustion.

[0154] Sequence information

[0155] Table 1: Information about the sequences involved in this invention is described in the table below: Detailed Implementation

[0156] The invention will now be described in the following non-limiting examples.

[0157] Those skilled in the art will understand that the embodiments are described by way of example only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0158] Example 1: Construction and Packaging of Anti-CD70 CAR

[0159] The CD70 antibody is a camel-derived nanobody (VHH) obtained in our laboratory through phage antibody screening technology (sequence shown in SEQ ID NOs:4, 7, and 11). Using CMV as the promoter (Addgene), the CAR construct was synthesized and cloned into the pCDH lentiviral vector (Addgene) backbone. Lentiviral vectors consisting of the antibody, CD8α hinge region, CD28 transmembrane domain, intracellular region of CD28 co-stimulatory signaling molecule, and CD3ζ signaling molecule were constructed and named CD70 CAR1, CD70 CAR2, and CD70 CAR3. The dual epitopes are two anti-CD70V antibodies. H Different combinations of H antibodies are linked together using (G4S)3, and named Tan-biCAR1-6. The sequences and compositions of the above anti-CD70 CARs are shown in Figure 1 and Table 2 below. The extracellular region of the negative control does not contain the antibody sequence, but the sequences and compositions of the other parts are the same as those of the above anti-CD70 CARs, and it is named Mock T.

[0160] Table 2: Sequence and composition of anti-CD70 CAR

[0161] After codon optimization of the designed fusion amino acid sequence using the GenSmart online tool, EcoRI and BamHI restriction sites were added to both segments, and the entire genome plasmid was synthesized by Anhui General Biotechnology (Systems) Co., Ltd. Then, lentivirus packaging was performed according to the following procedure:

[0162] (1) HEK 293T cells (ATCC) were digested with Tryple for 2 min, and then the digestion was terminated with medium containing 10% serum (Gibco, 10091148). The cells were centrifuged at 25°C for 5 min. After discarding the supernatant, the cells were washed once with PBS buffer and centrifuged at 25°C for 5 min.

[0163] (2) Resuspend the cells in 2 ml of culture medium containing 10% FBS (Gibco, 10091148) and perform cell counting. (The cell count is based on a concentration of 1.1 × 10⁻⁶ cells / mL.) 7 The number of cells was sufficient to ensure that the cells were evenly distributed in the T75 culture flask;

[0164] (3) The next day, the virus was packaged. The transfection reagent was prepared as follows: 40 μl PEI reagent (polyscience, 24765-1) + 460 μl serum-free DMEM (Gibco, 11965092), mixed well and allowed to stand for 5 min;

[0165] (4) Prepare DNA: mix the main plasmid (10 μl) + GP plasmid (7.5 μl) + VSVG plasmid (2.5 μl) thoroughly;

[0166] (5) Add the transfection reagent to the DNA, wash and mix well, and let stand at room temperature for 15-20 minutes.

[0167] (6) Add the suspension from step 5 to HEK 293T cells (cell density about 80%), mix well, and replace with 15 ml of DMEM (Gibco, 11965092) medium containing FBS after 4-6 hours.

[0168] (7) After 48 hours, collect the supernatant, store it in a 4°C refrigerator, and add 15 ml of preheated DMEM (Gibco, 11965092) medium containing 10% FBS.

[0169] (8) Collect the supernatant after 72 hours;

[0170] (9) Centrifuge at 1000g for 10 min, filter the supernatant through a 0.45μm filter membrane, and transfer it into an ultrafiltration tube;

[0171] (10) After centrifugation at 22000rpm for 2 hours at 4℃, discard the supernatant and resuspend the precipitate with 200μl of serum-free X-VIVO (LONZA, 04-418Q) to obtain concentrated virus supernatant.

[0172] Example 2: Construction and Validation of Anti-CD70 CAR-T

[0173] Peripheral blood mononuclear cells (PBMCs, Hycells, 5 × 10⁶) from a normal donor were used. 7 After being removed from the liquid nitrogen tank, cells were thawed in a 37°C water bath. The cell suspension was transferred to a 1.5 ml centrifuge tube, centrifuged at 500 × g for 5 min, and the cryopreservation solution was removed. The cells were resuspended in X-VIVO (LONZA, 04-418Q), centrifuged at 500 × g for 5 min, and the supernatant was discarded. T cells were isolated from the EasySep™ Human T Cell Isolation Kit using a negative selection enrichment and sorting kit (Stemcell, 17951).

[0174] (1) Add 50 μL of Isolation Cocktail and 800 μL of X-VIVO (LONZA, 04-418Q) medium and mix well;

[0175] (2) Add 1 ml of X-VIVO (LONZA, 04-418Q) medium to the cryovial and place it on a magnetic rack. Add the suspension from the above steps to the cryovial and mix well;

[0176] (3) Add 7 mL of culture medium to a 15 mL centrifuge tube, add cells from the cryopreservation tube, mix well, count the cells and centrifuge at 500×g for 5 min.

[0177] (4) Centrifuge again at 500×g for 5 min, then centrifuge at 1×10⁻⁶ g per centrifuge. 7 Each cell was resuspended in 100 μL of CD3 / CD28 activating magnetic beads (Gibco, 11161D) and 50 μL of culture medium.

[0178] (5) Place in an incubator for 7 minutes, take it out, mix it, and then place it back in the incubator. Repeat this process 3 times.

[0179] (6) According to every 2×10 6 The cell / ml culture medium ratio was used to culture cells in a T25 flask;

[0180] (7) On the second day after T cell sorting, take 1×10 6 Each cell was infected by adding 100 μL of the virus concentrate prepared in Example 1;

[0181] (8) Replace with fresh culture medium after 24 hours. CD4+ and CD8+ primary human T cells were cultured in X-VIVO medium (LONZA, 04-418Q) supplemented with 100 IU / ml IL-2 and 5% FBS, at a density of 2 × 10⁻⁶ cells / mL. 6 cell / ml;

[0182] (9) Subsequently, half the medium was changed every two days, and CAR-T cells cultured in vitro for 7 days were collected and washed with PBS. The cells were resuspended in PBS buffer (Procell, PB180327-500). Cells were stained with antibody (ACRO, CDL-HF249) to detect the CAR positivity rate of CAR-T cells in different treatment groups, and then analyzed using CytoFLEX S flow cytometer (Beckmancoulter). The analysis was performed on Kaluza 2.1 Flow Analysis Software (Beckmancoulter), and the results are shown in Figure 2. As can be seen from Figure 2, CAR-T cells expressing positive control CD70 CAR1, positive control CD70 CAR3, Tan-biCAR1-6 or negative control Mock T were successfully constructed and used in subsequent examples.

[0183] Example 3: Detection of CD70 expression in tumor cells

[0184] Human clear cell adenocarcinoma cells 786-0 and human glioma cells U251 were obtained from the ATCC cell bank and cultured in RPMI 1640 medium (Procell, PM150110) containing 10% serum (Gibco, 10091148). Human clear cell adenocarcinoma cells 786-0 and human glioma cells U251, after two passages of culture in vitro, were collected and washed with PBS (Procell, PB180327-500). The cells were resuspended in PBS buffer (Procell, PB180327-500). Cells were stained with an antibody (Biolegend, 355104). 5 × 10⁶ cells were collected. 5 Each tumor cell was incubated with 2 μL of Anti-Human CD70 antibody (Biolegend, 355104) at room temperature in the dark for 30 min. After washing three times with 1 mL of PBS buffer in the flow cytometry tube, the supernatant was discarded, and the cells were resuspended in 100 μL of PBS buffer to detect the expression level of CD70 on the cell surface. The analysis was then performed using a CytoFLEX S flow cytometer (Beckmancoulter). The analysis was performed on Kaluza 2.1 Flow Analysis Software (Beckmancoulter), and the results are shown in Figures 3 and 4. As can be seen from the figures, 786-0 and U251 cells highly expressed CD70.

[0185] Example 4: Killing effect of Anti CD70 CAR-T cells on 786-0-Luc cells

[0186] 786-0-Luc cells (ATCC) were cultured in vitro to quantitatively assess the cytotoxic function of CAR-T cells using a reporter gene assay. Cell density of cultured CAR-T cells and collected 786-0-Luc cells was determined by cell counting, and cell numbers were calculated based on cell density. Cytotoxicity was assessed by co-culturing dual-epitope CAR-T cells against 786-0-Luc cells in vitro. CAR-T cells and 786-0-Luc cells were co-incubated at effector-to-target ratios (E:T) of 1:3, 1:1, and 3:1 for 6 hours. Afterward, the cell culture plates were removed from the incubator and incubated at room temperature for 30 minutes to allow the temperature to equilibrate. 100 μL of One-Lite assay reagent (Vazyme, DD1203) was added to 100 μL of the cell culture. Cells were incubated at room temperature for at least 3 minutes to allow for complete lysis before assay. As shown in Figure 5, Tan-biCAR4 and Tan-biCAR6 exhibited tumor cell lysis rates comparable to or better than those of the positive controls (CD70 CAR1 and CD70 CAR3).

[0187] Example 5: Long-lasting killing effect of Anti CD70 CAR-T cells on 786-0 cells

[0188] CAR-T cells and 786-0 cells were co-cultured in vitro. CAR-T cells were resuspended in 100 μL of pre-warmed medium, and 786-0 cells were added at an E:T ratio of 3:1 to detect cell killing. The detection procedure was the same as described in Example 4. CAR-T cells were subjected to four rounds of antigen stimulation. CAR-T cells were resuspended in 2 mL of pre-warmed medium, and 786-0 cells were added at an E:T ratio of 2:1. After co-culturing for 16 h, the cells were centrifuged at 500 g for 5 min to remove the old medium, and the CAR-T cells were resuspended in fresh medium. The above operation was repeated three times after 48 h, and CAR-T cells were detected after each round of stimulation. The results are shown in Figures 6-12. Figure 6 shows that the positivity rate of CAR-T cells gradually increased, and Figure 7 shows the cell killing effect of the example dual-epitope CAR-T cells after four rounds of antigen stimulation. The Tan-biCAR4 sequence exhibits superior cytotoxicity. Figures 8 and 9 show the exhaustion characterization of the example dual-epitope CAR-T cells after four rounds of antigen stimulation. The Tan-biCAR4 sequence maintains high cytotoxicity while exhibiting lower fatigue. Figures 10-12 show the results of in vitro IFNγ, TNF-α, and IL-2 release assays of T cells containing the exemplary dual-epitope CD70 CAR against 786-0-Luc cells.

[0189] Example 6: Killing effect of Anti CD70 CAR-T cells on U251-Luc cells

[0190] U251-Luc cells (ATCC) were cultured in vitro to quantitatively assess the cytotoxic function of CAR-T cells using a reporter gene assay. Cell density of cultured CAR-T cells and collected U251-Luc cells was determined by cell counting, and cell numbers were calculated based on cell density. Cytotoxicity was assessed by co-culturing dual-epitope CAR-T cells against U251-Luc cells in vitro. CAR-T cells and U251-Luc cells were co-incubated at effector-to-target ratios (E:T) of 16:1, 8:1, 4:1, and 2:1 for 6 hours. Afterward, the cell culture plates were removed from the incubator and incubated at room temperature for 30 minutes to allow the temperature to equilibrate. 100 μL of One-Lite assay reagent (Vazyme, DD1203) was added to 100 μL of the cell culture. Cells were incubated at room temperature for at least 3 minutes to allow for complete lysis before assay. As shown in Figure 13, Tan-biCAR4 exhibited a tumor cell lysis rate comparable to or better than that of the positive controls (CD70 CAR1 and CD70 CAR3).

[0191] Example 7: Long-lasting killing effect of Anti CD70 CAR-T cells on U251 cells

[0192] CAR-T cells and U251 cells were co-cultured in vitro. CAR-T cells were resuspended in 100 μL of pre-warmed medium, and U251 cells were added at E:T ratios of 16:1, 8:1, 4:1, and 2:1 to detect cell killing. The detection procedure was the same as described in Example 6. CAR-T cells were subjected to four rounds of antigen stimulation. CAR-T cells were resuspended in 2 mL of pre-warmed medium, and U251 cells were added at an E:T ratio of 2:1. After co-culturing for 16 h, the cells were centrifuged at 500 g for 5 min to remove the old medium, and the CAR-T cells were resuspended in fresh medium. The above operation was repeated three times after 48 h, and the CAR-T cells were detected after each round of stimulation. The results are shown in Figures 14-17. Figure 14 shows that Tan-biCAR4 has the strongest killing ability against U251-LUC cells. Figure 15 shows that after four rounds of antigen stimulation, Tan-biCAR4 sequence CAR-T cells exhibit superior cell proliferation ability. Figure 16 shows that under the re-stimulation of antigen, exemplary dual-episode CAR-T cells (CAR-T cells expressing Tan-biCAR3 and Tan-biCAR4, respectively) can continue to generate a large number of cloned effector memory T cells (Tem) carrying the same antigen. Figure 17 shows that compared with the more cytotoxic single-episode CAR, the dual-episode CAR Tan-biCAR4 of CD70-3×CD70-1 combination has lower exhaustion.

[0193] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. Chimeric antigen receptor, which includes an extracellular antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signal transduction domain; The extracellular antigen-binding domain includes a first antigen-binding domain targeting CD70 and a second antigen-binding domain targeting CD70, wherein the first antigen-binding domain and the second antigen-binding domain target different epitopes of CD70. The first antigen-binding domain and the second antigen-binding domain are nanobodies, wherein, (1) The first antigen-binding domain includes CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3, and the second antigen-binding domain includes CDR1 as shown in SEQ ID NO:8, CDR2 as shown in SEQ ID NO:9, and CDR3 as shown in SEQ ID NO:

10. (2) The first antigen-binding domain includes CDR1 as shown in SEQ ID NO:8, CDR2 as shown in SEQ ID NO:9, and CDR3 as shown in SEQ ID NO:10, and the second antigen-binding domain includes CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:

3. (3) The first antigen-binding domain includes CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3, and the second antigen-binding domain includes CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:6; (4) The first antigen-binding domain includes CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:6, and the second antigen-binding domain includes CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3; (5) The first antigen-binding domain comprises CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:6, and the second antigen-binding domain comprises CDR1 as shown in SEQ ID NO:8, CDR2 as shown in SEQ ID NO:9, and CDR3 as shown in SEQ ID NO:10; or (6) The first antigen-binding domain includes CDR1 as shown in SEQ ID NO:8, CDR2 as shown in SEQ ID NO:9, and CDR3 as shown in SEQ ID NO:10, and the second antigen-binding domain includes CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:

6.

2. The chimeric antigen receptor of claim 1, wherein, (1) The first antigen-binding domain comprises a nanobody as shown in SEQ ID NO:4 or a variant thereof, and the second antigen-binding domain comprises a nanobody as shown in SEQ ID NO:11 or a variant thereof; (2) The first antigen-binding domain comprises a nanobody as shown in SEQ ID NO:11 or a variant thereof, and the second antigen-binding domain comprises a nanobody as shown in SEQ ID NO:4 or a variant thereof; (3) The first antigen-binding domain comprises a nanobody as shown in SEQ ID NO:4 or a variant thereof, and the second antigen-binding domain comprises a nanobody as shown in SEQ ID NO:7 or a variant thereof; (4) The first antigen-binding domain comprises a nanobody as shown in SEQ ID NO:7 or a variant thereof, and the second antigen-binding domain comprises a nanobody as shown in SEQ ID NO:4 or a variant thereof; (5) The first antigen-binding domain comprises a nanobody as shown in SEQ ID NO:7 or a variant thereof, and the second antigen-binding domain comprises a nanobody as shown in SEQ ID NO:11 or a variant thereof; or (6) The first antigen-binding domain comprises a nanobody as shown in SEQ ID NO:11 or a variant thereof, and the second antigen-binding domain comprises a nanobody as shown in SEQ ID NO:7 or a variant thereof; The variant has at least 80% sequence identity with the sequence from which it originates, or has one or more amino acid substitutions, deletions, or additions compared to the original sequence; preferably, the substitutions are conservative substitutions. Preferably, (1) the first antigen-binding domain comprises a nanobody as shown in SEQ ID NO:4, and the second antigen-binding domain comprises a nanobody as shown in SEQ ID NO:11; (2) The first antigen-binding domain comprises a nanobody as shown in SEQ ID NO:11, and the second antigen-binding domain comprises a nanobody as shown in SEQ ID NO:4; (3) The first antigen-binding domain comprises a nanobody as shown in SEQ ID NO:4, and the second antigen-binding domain comprises a nanobody as shown in SEQ ID NO:7; (4) The first antigen-binding domain comprises a nanobody as shown in SEQ ID NO:7, and the second antigen-binding domain comprises a nanobody as shown in SEQ ID NO:4; (5) The first antigen-binding domain comprises a nanobody as shown in SEQ ID NO:7, and the second antigen-binding domain comprises a nanobody as shown in SEQ ID NO:11; or (6) The first antigen-binding domain contains a nanobody as shown in SEQ ID NO:11, and the second antigen-binding domain contains a nanobody as shown in SEQ ID NO:

7.

3. The chimeric antigen receptor according to claim 1 or 2, wherein, The first antigen-binding domain is directly or via a peptide linker connected to the N-terminus or C-terminus of the second antigen-binding domain; Preferably, the peptide linker is (GmS)n, where m and n are independently integers not less than 0, for example, independently 1, 2, 3 or 4; Preferably, the extracellular antigen-binding domain comprises the sequence shown in any one of SEQ ID NOs:12-17.

4. The chimeric antigen receptor according to any one of claims 1-3, further comprising a signal peptide at its N-terminus; Preferably, the signal peptide is derived from CD8α, GM-CSF receptor α, or IgG1 heavy chain; Preferably, the signal peptide comprises the sequence shown in SEQ ID NO:

28.

5. The chimeric antigen receptor according to any one of claims 1-4, wherein, The spacer region is selected from the hinge region and / or the CH2 and CH3 regions of immunoglobulins (e.g., IgG1 or IgG4); Preferably, the spacer region structural domain includes the hinge region of CD8α; Preferably, the spacer region structure domain comprises the sequence shown in SEQ ID NO:

24.

6. The chimeric antigen receptor according to any one of claims 1-5, wherein, The transmembrane domain is selected from the transmembrane regions of the following proteins: CD8α, CD4, CD28, CD137, CD80, CD86, CD152, PD1; Preferably, the transmembrane domain comprises the sequence shown in SEQ ID NO:

25.

7. The chimeric antigen receptor according to any one of claims 1-6, wherein, The intracellular signal transduction domain includes a co-stimulatory signal transduction domain; Preferably, the co-stimulatory signal transduction domain comprises intracellular signal transduction domains selected from the following proteins: ligands of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA 1, ICOS, CD2, CD7, LIGHT, NKG2C, B7H3, CD83, and combinations thereof; Preferably, the co-stimulatory signal transduction domain includes an intracellular signal transduction domain of CD28 and / or an intracellular signal transduction domain of CD137; Preferably, the co-stimulatory signal transduction domain comprises the sequence shown in SEQ ID NO:

27.

8. The chimeric antigen receptor according to any one of claims 1-7, wherein, The intracellular signal transduction domain includes a primary signal transduction domain; Preferably, the primary signal transduction domain is derived from CD3ζ; Preferably, the primary signal transduction domain comprises the sequence shown in SEQ ID NO:

26.

9. The chimeric antigen receptor according to any one of claims 1-8, wherein, The chimeric antigen receptor comprises, from its N-terminus to its C-terminus, the signal peptide, the extracellular antigen-binding domain, the spacer domain, the transmembrane domain, and the intracellular signal transduction domain. Preferably, the signal peptide is derived from CD8α; Preferably, the spacer structure domain includes the hinge region of CD8α; Preferably, the transmembrane structural domain includes the transmembrane region of CD28; Preferably, the intracellular signal transduction domain includes a co-stimulatory signal transduction domain and a primary signal transduction domain, the co-stimulatory signal transduction domain being connected to the N-terminus of the primary signal transduction domain, the co-stimulatory signal transduction domain including the intracellular signal transduction domain of CD28, and the primary signal transduction domain originating from CD3ζ; Preferably, the chimeric antigen receptor comprises the sequence shown in any one of SEQ ID NOs:18-23.

10. An isolated nucleic acid molecule encoding the chimeric antigen receptor as described in any one of claims 1-9.

11. A vector comprising the isolated nucleic acid molecule of claim 10; preferably, the vector is a cloning vector or an expression vector.

12. An engineered immune cell comprising the chimeric antigen receptor of any one of claims 1-9, the isolated nucleic acid molecule of claim 10, or the vector of claim 11; Preferably, the engineered immune cells are human immune cells; Preferably, the engineered immune cells are selected from T cells, NK cells, macrophages, peripheral blood mononuclear cells, hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

13. A pharmaceutical composition comprising the chimeric antigen receptor of any one of claims 1-9, the isolated nucleic acid molecule of claim 10, the carrier of claim 11, or the engineered immune cell of claim 12; and pharmaceutically acceptable excipients; Preferably, the pharmaceutical composition further comprises an additional antitumor drug.

14. The use of the chimeric antigen receptor of any one of claims 1-9, the isolated nucleic acid molecule of claim 10, the carrier of claim 11, the engineered immune cell of claim 12, or the pharmaceutical composition of claim 13 in the preparation of a medicament for the treatment and / or prevention of tumors or autoimmune diseases; Preferably, the tumor is a CD70-positive tumor; Preferably, the tumor is a solid tumor or a hematogenous tumor; Preferably, the solid tumor is selected from renal cell carcinoma, lung cancer, glioma, nasopharyngeal carcinoma, gastric cancer, head and neck cancer, skin cancer, colorectal cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, colorectal cancer, or oral squamous cell carcinoma. Preferably, the hematogenous tumor is selected from Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, or leukemia; Preferably, the autoimmune disease is selected from arthritis, rheumatoid arthritis, psoriatic arthritis, lupus, lupus nephritis, systemic lupus erythematosus, psoriasis, vitiligo, alopecia areata, inflammatory bowel disease, ulcerative colitis, Crohn's disease, type I diabetes, multiple sclerosis, autoimmune hepatitis, primary biliary cirrhosis, celiac disease, scleroderma, Graves' disease, Hashimoto's thyroiditis, ankylosing spondylitis, myasthenia gravis, Sjögren's syndrome, IgA nephropathy, IgG4-related disease, vasculitis, ANCA-associated vasculitis, uveitis, pemphigus, bullous pemphigoid, or autoimmune hemolytic anemia; Preferably, the subject is a mammal, such as a human; Preferably, the chimeric antigen receptor, isolated nucleic acid molecule, carrier, engineered immune cell, or pharmaceutical composition is used alone or in combination with other antitumor drugs.

15. A method for treating and / or preventing CD70-related diseases, comprising administering to a subject in need of the chimeric antigen receptor of any one of claims 1-9, the isolated nucleic acid molecule of claim 10, the carrier of claim 11, the engineered immune cells of claim 12, or the pharmaceutical composition of claim 13, the steps of which are: The diseases associated with CD70 are tumors or autoimmune diseases; Preferably, the tumor is a CD70-positive tumor; Preferably, the tumor is a solid tumor or a hematogenous tumor; Preferably, the solid tumor is selected from renal cell carcinoma, lung cancer, glioma, nasopharyngeal carcinoma, gastric cancer, head and neck cancer, skin cancer, colorectal cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, colorectal cancer, or oral squamous cell carcinoma. Preferably, the hematogenous tumor is selected from Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, or leukemia; Preferably, the autoimmune disease is selected from arthritis, rheumatoid arthritis, psoriatic arthritis, lupus, lupus nephritis, systemic lupus erythematosus, psoriasis, vitiligo, alopecia areata, inflammatory bowel disease, ulcerative colitis, Crohn's disease, type I diabetes, multiple sclerosis, autoimmune hepatitis, primary biliary cirrhosis, celiac disease, scleroderma, Graves' disease, Hashimoto's thyroiditis, ankylosing spondylitis, myasthenia gravis, Sjögren's syndrome, IgA nephropathy, IgG4-related disease, vasculitis, ANCA-associated vasculitis, uveitis, pemphigus, bullous pemphigoid, or autoimmune hemolytic anemia; Preferably, the subject is a mammal, such as a human; Preferably, the chimeric antigen receptor, isolated nucleic acid molecule, carrier, engineered immune cell, or pharmaceutical composition is used alone or in combination with other antitumor drugs.

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