Therapeutic immune cell

Abstract

The present invention relates to the field of biomedicine, and in particular to the field of cell therapy. More specifically, provided is an improved therapeutic immune cell, such as a T cell, which co-expresses a synthetic T-cell receptor and antigen receptor (STAR) and membrane-bound IL-15 (mbIL-15). Also provided are a vector and method for preparing the therapeutic immune cell.

Description

Therapeutic immune cells Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the field of cell therapy. More specifically, this invention provides an improved therapeutic immune cell, such as a T cell, which co-expresses a synthetic T-cell receptor and antigen receptor (STAR) and membrane-bound IL-15 (mbIL-15). This invention also provides a vector and method for preparing said therapeutic immune cells. Background Technology

[0002] In recent years, cellular immunotherapy has shown good efficacy in the treatment of hematological malignancies such as leukemia and lymphoma. However, in the treatment of solid tumors, due to factors such as the immune escape mechanisms of solid tumors and the suppression of the tumor microenvironment, the function of CAR-T cells is suppressed in the tumor microenvironment, and T cells are prone to exhaustion and apoptosis. Recent studies have shown that this T cell dysfunction may be related to the nature of the chimeric antigen receptor signaling pathway.

[0003] WO2021135178A1 discloses that the natural T-cell receptor (TCR) complex contains 10 ITAM signaling sequences, theoretically capable of transmitting a stronger signal than CAR. Studies have shown that while TCR signaling is slower than CAR signaling, it is more persistent. Therefore, utilizing the signal transduction function of the natural TCR, it is possible to construct a novel receptor to alleviate T-cell dysfunction and enable it to better exert its anti-solid tumor effects. The extracellular region of the TCR is very similar to the Fab domain of an antibody; therefore, the variable region sequence of the TCR can be replaced with the variable region sequence of an antibody, resulting in a synthetic T-cell receptor antigen receptor (STAR). This STAR possesses both the specificity of an antibody and the superior signal transduction function of the natural TCR, and can mediate complete T-cell activation. Therefore, the synthetic T-cell receptor antigen receptor (STAR) can alleviate T-cell dysfunction and enable it to better exert its anti-solid tumor effects.

[0004] Existing literature (Dwyer CJ et al. (2019) Fueling Cancer Immunotherapy With Common Gamma Chain Cytokines. Front. Immunol. 10:263) reports that membrane-bound IL-15 protein is mainly produced by dendritic cells (DCs), monocytes, and macrophages. It can stimulate the activation and proliferation of T cells and NK cells, support the proliferation of CD8+ T cells with a long lifespan memory phenotype, inhibit IL-2-induced AICD, and maintain a higher proportion of Tscm memory cells with lower differentiation and stronger proliferative and survival capabilities. IL-15 belongs to the γ-chain fusion protein family and shares IL2Rβ and IL-2Rγ with IL-2, with similar downstream signaling. IL-15Rα is a receptor specific to IL-15, with an affinity 1000 times higher than IL-2Rα (10^6). -11 IL-15 can be maintained on the surface of APC cells, thereby activating effector cells. Previous literature (Hurton LV et al. (2016) Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells. Proc Natl Acad Sci US A. 113(48):E7788-E7797) has reported that CAR-T cells can enhance their persistence and proliferation by co-expressing membrane-bound IL-15. However, the effects of membrane-bound IL-15 on other T cells are unknown. Summary of the Invention

[0005] In one aspect, the present invention provides an isolated therapeutic immune cell comprising a synthetic T-cell receptor antigen receptor (STAR) and a membrane-bound IL-15 protein (mbIL-15).

[0006] In another aspect, the present invention provides an expression vector comprising the coding sequence of the synthetic T-cell receptor antigen receptor (STAR) described herein and the coding sequence of the membrane-bound IL-15 protein (mbIL-15) described herein.

[0007] In another aspect, the present invention provides a method for preparing therapeutic immune cells, comprising:

[0008] Step 1) Provide initial immune cells;

[0009] Step 2) Introduce the expression vector of the present invention into the initiating immune cells; and

[0010] Step 3) Harvest the immune cells obtained in Step 2).

[0011] In another aspect, the present invention provides a therapeutic immune cell, such as a T cell, that comprises the expression vector of the present invention, or that can be obtained or acquired by the expression vector of the present invention or by the method of the present invention.

[0012] In another aspect, the present invention provides a pharmaceutical composition comprising the therapeutic immune cells of the present invention and / or the expression vector of the present invention, and a pharmaceutically acceptable carrier, preferably for use in treating a disease in a subject.

[0013] In another aspect, the present invention provides the use of the therapeutic immune cells of the present invention, the expression vectors of the present invention, and / or the pharmaceutical compositions of the present invention in the preparation of medicaments for treating diseases in subjects.

[0014] In another aspect, the present invention provides a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the therapeutic immune cells of the present invention, the expression vector of the present invention, and / or a pharmaceutical composition of the present invention. Attached Figure Description

[0015] Figure 1. Schematic diagram of the vector structure used to co-express different proteins and MSLN STAR.

[0016] Figure 2. Results of protein surface assay in MSLN STAR-T cells co-expressing different proteins.

[0017] Figure 3. Results of functional protein expression detection in MSLN STAR-T cells co-expressing different proteins. A: mbIL-15 protein expression detection results in MSLN STAR-T cells; B: sIL-15 protein expression detection results in MSLN STAR-T cells; C: IL-10 protein expression detection results in MSLN STAR-T cells; D: p52 protein expression detection results in MSLN STAR-T cells.

[0018] Figure 4. Results of the killing efficiency of MSLN STAR-T cells co-expressing different proteins against different target cells. A shows the killing efficiency of MSLN STAR-T cells co-expressing different proteins against SKOV3 target cells; B shows the killing efficiency of MSLN STAR-T cells co-expressing different proteins against Aspc-1 target cells.

[0019] Figure 5. Killing of tumor cells in vivo by MSLN STAR-T cells co-expressing different proteins.

[0020] Figure 6. Amplification detection in an in vivo tumor-bearing model of MSLN STAR-T co-expressing different proteins.

[0021] Figure 7. Structure of GC33-STAR expression vector co-expressing mbIL-15 or CXCR2, using both HLTV and MND promoters.

[0022] Figure 8. Upper membrane expression and detection of GPC3-STAR.

[0023] Figure 9. In vitro continuous killing function of GPC3-STAR-T cells co-expressing different proteins driven by different promoters.

[0024] Figure 10. Cytokine secretion in GPC3-STAR-T cells co-expressing different proteins driven by different promoters.

[0025] Figure 11. In vivo efficacy validation of GPC3 STAR-T co-expressing mbIL-15.

[0026] Figure 12. Viability changes of different samples under the same culture conditions.

[0027] Figure 13. Changes in pSTAT5 expression levels before and after incubation of different samples with Huh-7 cells.

[0028] Figure 14. Schematic diagram of the vector structure for co-expressing mbIL-15 with the dual epitope LILRB4 STAR.

[0029] Figure 15. In vitro killing of tumor cells by LILRB4 STAR-T cells co-expressing mbIL-15.

[0030] Figure 16. In vivo killing of tumor cells by LILRB4 STAR-T cells co-expressing mbIL-15.

[0031] Figure 17. Amplification detection of LILRB4 STAR-T in vivo tumor-bearing model co-expressing mbIL-15 dual epitopes.

[0032] Figure 18. Viability changes of different samples under the same culture conditions.

[0033] Figure 19. Changes in pSTAT5 expression levels before and after incubation of different samples with MV-4-11 cells.

[0034] Figure 20. Vector structures for co-expressing mbIL-15 and NBC11-NLB14 STAR, and vector structures for expressing NBC11-NLB14 STAR.

[0035] Figure 21. In vitro upper membrane expression of NBC11-NLB14 STAR-mbIL-15.

[0036] Figure 22. Expansion of NBC11-NLB14 STAR-T cells under NCI-H929 target cell stimulation (E:T = 0.8:1).

[0037] Figure 23. Detection of continuous killing efficiency of T cells expressing NBC11-NLB14 STAR-mbIL-15.

[0038] Figure 24. In vivo efficacy of T cells expressing NBC11-NLB14 STAR-mbIL-15 in a mixed tumor model. Detailed Implementation

[0039] definition

[0040] Unless otherwise indicated or defined, all terms used herein have their ordinary meaning as will be understood by those skilled in the art. References include, for example, standard manuals such as Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd edition), Volumes 1–3, Cold Spring Harbor Laboratory Press (1989); Lewin, “Genes IV”, Oxford University Press, New York (1990); and Roitt et al., “Immunology” (2nd edition), Gower Medical Publishing, London, New York (1989), and general prior art cited herein; furthermore, unless otherwise stated, all methods, procedures, techniques, and operations not specifically detailed herein can and have been performed in a manner known per se as will be understood by those skilled in the art. References also include, for example, standard manuals, the aforementioned general prior art, and other references cited therein. All references described herein are incorporated herein by reference in their entirety.

[0041] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0042] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the stated sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still possessing the activities described in this invention. Furthermore, those skilled in the art will understand that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in certain practical situations (e.g., when expressed in a specific expression system) without substantially affecting the polypeptide's function. Therefore, when describing a specific polypeptide amino acid sequence in this specification and claims, although it may not contain the methionine encoded by the start codon at the N-terminus, the sequence containing that methionine is still included, and correspondingly, its encoding nucleotide sequence may also contain the start codon; and vice versa.

[0043] A polypeptide or nucleic acid molecule is considered "isolated" when it has been separated from at least one other component (e.g., another protein / peptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or trace component) that is normally associated with it in that source or medium (culture medium), compared to its natural biological source and / or the reaction medium or culture medium from which the molecule is obtained. Specifically, a polypeptide or nucleic acid molecule is considered "isolated" when it has been purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold, and up to 1000-fold or more. "Isolated" polypeptide or nucleic acid molecules are preferably substantially homogeneous, as determined by suitable techniques (e.g., suitable chromatographic techniques, such as polyacrylamide gel electrophoresis).

[0044] As used in this article, the synthetic T-cell receptor antigen receptor (STAR) refers to a modified TCR in which the variable region sequence of the TCR is replaced with an antibody variable region sequence or other receptor sequences, while the constant region sequence of the TCR can also be modified.

[0045] As used herein, an "antigen-binding region" (e.g., an antigen-binding region in STAR) means that it can specifically bind to a target antigen, either alone or in combination with another antigen-binding region. The antigen-binding region can be derived from an antibody that specifically binds to the target antigen, including any commercially available antibody. An antigen-binding region can also be derived from a receptor that binds to a specific target protein.

[0046] As used herein, “antibody” refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetic (e.g., recombinant), including any fragment that retains the binding specificity of the full-length immunoglobulin molecule, containing at least a portion of the variable region of the immunoglobulin molecule. Therefore, antibodies include any protein having a binding domain homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). Antibodies include antibody fragments. As used herein, the term antibody includes synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, camel antibodies, single-domain antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab′ fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), biantibodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The antibodies described herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0047] As used herein, a variable domain or variable region is a specific Ig domain of the antibody heavy or light chain, containing a variable amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has a variable region VL (also denoted as V). L ) and VH (or also represented as V) L Variable domains provide antigen specificity and are therefore responsible for antigen recognition. Each variable region contains a CDR and a frame region (FR), with the CDR being part of the antigen-binding site.

[0048] As used herein, “hypervariant region,” “HV,” “complementarity-determining region,” and “CDR” and “antibody CDR” are interchangeably used to refer to one of the multiple portions within each variable region that together form the antigen-binding site of an antibody. Each variable region contains three CDRs, named CDR1, CDR2, and CDR3. For example, for a conventional four-chain antibody, the light chain variable region contains three CDRs, named VL CDR1, VL CDR2, and VL CDR3 (or LCDR1, LCDR2, and LCDR3); the heavy chain variable region contains three CDRs, named VH CDR1, VH CDR2, and VH CDR3 (or HCDR1, HCDR2, and HCDR3). For camel antibodies or single-domain antibodies, since they have only one variable region, they contain only three CDRs, named CDR1, CDR2, and CDR3.

[0049] In the context of this invention, the terms "single-domain antibody", "nanobody", "heavy chain single-domain antibody", "VHH", "VHH domain", "VHH antibody fragment", and "VHH antibody" are used interchangeably.

[0050] A “single-domain antibody” is a variable domain of an antigen-binding immunoglobulin called a “heavy-chain antibody” (i.e., an antibody lacking a light chain) (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)). The term “VHH” is used to distinguish the variable region of a heavy-chain antibody from the heavy-chain variable region (referred to herein as “VH”) present in conventional 4-chain antibodies and the light-chain variable region (referred herein as “VL”) present in conventional 4-chain antibodies. A VHH specifically binds to an epitope without the need for other antigen-binding domains (as opposed to the VH or VL in conventional 4-chain antibodies, where the epitope is recognized by both VL and VH). A VHH is a small, stable, and highly efficient antigen-recognizing unit formed by a single domain.

[0051] As used herein, "amino acid number reference SEQ ID NO:x" (SEQ ID NO:x being a specific sequence listed herein) refers to the position number of the described specific amino acid being the position number of the corresponding amino acid in SEQ ID NO:x. The correspondence between amino acids in different sequences can be determined using sequence alignment methods known in the art. For example, amino acid correspondence can be determined using the EMBL-EBI online alignment tool (https: / / www.ebi.ac.uk / Tools / psa / ), where two sequences can be aligned using the Needleman-Wunsch algorithm with default parameters. For example, if the alanine at position 46 from the N-terminus of a polypeptide is aligned with the 48th amino acid in SEQ ID NO:x in sequence alignment, then this amino acid in the polypeptide can also be described herein as "the alanine at position 48 of the polypeptide, the position of which is referenced to SEQ ID NO:x".

[0052] The proteins / peptides mentioned in this invention may contain a signal peptide (or guide sequence) at their N-terminus. Those skilled in the art will understand that in cells, the signal peptide sequence can guide the protein / peptide to a specific location on the cell membrane, and it may itself be cleaved and not included in the final product. Exemplary signal peptides include, but are not limited to, IgE signal peptide, GM-CSF signal peptide, bovine prolactin pre-signal peptide, and natural signal peptides of the mentioned proteins / peptides such as IL-15Ra signal peptide, IL-15 signal peptide, etc. These signal peptide sequences are known in the art or can be readily identified by those skilled in the art based on existing knowledge in the art.

[0053] The "expression vector" of the present invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or a translatable RNA (such as mRNA). In some preferred embodiments, the expression vector is a viral vector, such as a lentiviral vector.

[0054] As used herein, the term "operably linked" refers to the linking of an expression regulatory element (e.g., but not limited to, promoter sequences, transcription termination sequences, etc.) to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) such that transcription of the nucleotide sequence is controlled and regulated by the transcription regulatory element. Techniques for operably linking regulatory element regions to nucleic acid molecules are known in the art. The terms "regulatory sequence" and "regulatory element" are used interchangeably, referring to a nucleotide sequence located upstream (5′ non-coding sequence), midway, or downstream (3′ non-coding sequence) of a coding sequence and influencing transcription, RNA processing, or stability or translation of the relevant coding sequence. An expression regulatory element refers to a nucleotide sequence capable of controlling transcription, RNA processing, or stability or translation of a nucleotide sequence of interest. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, enhancers, and polyadenylation recognition sequences. Suitable promoters include, but are not limited to, the PGK promoter, hEF1a promoter, HTLV promoter, and MND promoter.

[0055] As used herein, "object" refers to an organism that suffers from or is susceptible to a disease (such as cancer) that can be treated by the antibodies, cells, methods, or pharmaceutical compositions of the present invention. Non-limiting examples include humans, cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals. In a preferred embodiment, the object is a human.

[0056] The term "pharmaceutically acceptable carrier" as used herein includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[0057] As used herein, "therapeutic effective amount" or "therapeutic effective dose" or "effective amount" refers to an amount of substance, compound, material, or cell that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition. For example, an "effective amount" of the cellular or pharmaceutical composition of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of damage or disability caused by disease suffering. For example, in the treatment of tumors, an "effective amount" of the antibody, cell, expression vector, or pharmaceutical composition of the present invention preferably inhibits tumor cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system for predicting the efficacy against human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit tumor cell growth, which can be determined in vitro by tests known to those skilled in the art.

[0058] Therapeutic immune cells

[0059] In one aspect, the present invention provides an isolated therapeutic immune cell comprising a synthetic T-cell receptor antigen receptor (STAR) and a membrane-bound IL-15 protein (mbIL-15). In some embodiments, the therapeutic immune cell co-expresses STAR and mbIL-15.

[0060] In this document, mbIL-15 or mbIL15 refers to a fusion protein formed by linking IL-15 with the extracellular domain of IL-15Ra (e.g., via a linker). An exemplary amino acid sequence of IL-15 is shown in SEQ ID NO:18, but also includes amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, or even at least 99% sequence identity with SEQ ID NO:18. An exemplary amino acid sequence of the extracellular domain of IL-15Ra is shown in SEQ ID NO:19, but also includes amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, or even at least 99% sequence identity with SEQ ID NO:19. An exemplary amino acid sequence of the linker connecting the extracellular domain of IL-15Ra to IL-15 is shown in SEQ ID NO:20. An exemplary amino acid sequence of mbIL-15 is shown in SEQ ID NO:21, but also includes amino acid sequences that have at least 80%, at least 85%, at least 90%, at least 95%, or even at least 99% sequence identity with SEQ ID NO:21.

[0061] In some embodiments, the STAR comprises an α chain and a β chain, the α chain comprising a first constant region, the β chain comprising a second constant region, and wherein the α chain and / or the β chain further comprises an antigen-binding region that specifically binds to the target antigen.

[0062] In some embodiments, the first constant region is a natural TCRα chain constant region, such as a natural human TCRα chain constant region or a natural mouse TCRα chain constant region. An exemplary natural human TCRα chain constant region comprises the amino acid sequence shown in SEQ ID NO:1. An exemplary natural mouse TCRα chain constant region comprises the amino acid sequence shown in SEQ ID NO:2.

[0063] In some implementations, the first constant region is a modified TCRα chain constant region.

[0064] In some embodiments, the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, wherein the amino acid at position 48, for example threonine T, is mutated to cysteine ​​C, relative to the wild-type mouse TCRα chain constant region.

[0065] In some embodiments, the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, wherein, relative to the wild-type mouse TCRα chain constant region, the amino acid at position 112, such as serine S, is replaced with leucine L; the amino acid at position 114, such as methionine M, is replaced with isoleucine I; and the amino acid at position 115, such as glycine G, is replaced with valine V.

[0066] In some embodiments, the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, which, relative to the wild-type mouse TCRα chain constant region, has its 6th amino acid, such as E, replaced by D, its 13th K replaced by R, and its 15th-18th amino acids deleted.

[0067] In some embodiments, the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, which, relative to the wild-type mouse TCRα chain constant region, has the following modifications: the amino acid at position 48, such as threonine (T), is mutated to cysteine ​​(C); the amino acid at position 112, such as serine (S), is mutated to leucine (L); the amino acid at position 114, such as methionine (M), is mutated to isoleucine (I); and the amino acid at position 115, such as glycine (G), is mutated to valine (V).

[0068] In some embodiments, the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, which, relative to the wild-type mouse TCRα chain constant region, has the following modifications: amino acid at position 6, such as E, is replaced by D; amino acid at position 13, K, is replaced by R; amino acids at positions 15-18 are deleted; amino acid at position 48, such as threonine (T), is mutated to cysteine ​​(C); amino acid at position 112, such as serine (S), is replaced by leucine (L); amino acid at position 114, such as methionine (M), is replaced by isoleucine (I); and amino acid at position 115, such as glycine (G), is replaced by valine (V).

[0069] In some embodiments, the TCRα chain constant region is a non-intracellular region relative to the wild-type TCRα chain constant region, for example, the non-intracellular region of the constant region is missing amino acids 136-137.

[0070] In some embodiments, the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, which, relative to the wild-type mouse TCRα chain constant region, lacks the intracellular region of the constant region, for example, the deletion of amino acids 136-137.

[0071] In some specific embodiments, the modified TCRα chain constant region contains an amino acid sequence shown in one of SEQ ID NO:3-7.

[0072] In some embodiments, the second constant region is a natural TCRβ chain constant region, such as a natural human TCRβ chain constant region or a natural mouse TCRβ chain constant region. An exemplary natural human TCRβ chain constant region comprises the amino acid sequence shown in SEQ ID NO:8. An exemplary natural mouse TCRβ chain constant region comprises the amino acid sequence shown in SEQ ID NO:9.

[0073] In some implementations, the second constant region is a modified TCRβ chain constant region.

[0074] In some embodiments, the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, wherein the amino acid at position 56, for example serine S, is mutated to cysteine ​​C, relative to the wild-type mouse TCRβ chain constant region.

[0075] In some embodiments, the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, which, relative to the wild-type mouse TCRβ chain constant region, has the following modifications: the amino acid at position 3, such as R, is replaced by K; the amino acid at position 6, such as T, is replaced by F; the amino acid at position 9, such as K, is replaced by E; the amino acid at position 11, such as S, is replaced by A; the amino acid at position 12, such as L, is replaced by V; and the amino acids at positions 17 and 21-25 are deleted.

[0076] In some embodiments, the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, which, relative to the wild-type mouse TCRβ chain constant region, has the following modifications: the amino acid at position 56, such as serine (S), is mutated to cysteine ​​(C); the amino acid at position 3, such as R, is replaced by K; the amino acid at position 6, such as T, is replaced by F; the amino acid at position 9, such as K, is replaced by E; the amino acid at position 11, such as S, is replaced by A; the amino acid at position 12, such as L, is replaced by V; and the amino acids at positions 17 and 21-25 are deleted.

[0077] In some embodiments, the TCRβ chain constant region is a non-intracellular region relative to the wild-type TCRβ chain constant region, for example, the intracellular region of the constant region is missing amino acids 167-172.

[0078] In some embodiments, the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, which, relative to the wild-type mouse TCRβ chain constant region, lacks the intracellular region of the constant region, for example, the deletion of amino acids 167-172.

[0079] In some specific embodiments, the modified TCRβ chain constant region contains an amino acid sequence shown in one of SEQ ID NO:10-14.

[0080] In some embodiments, the first constant region comprises the amino acid sequence shown in SEQ ID NO:3, and the second constant region comprises the amino acid sequence shown in SEQ ID NO:10. In some embodiments, the first constant region comprises the amino acid sequence shown in SEQ ID NO:6, and the second constant region comprises the amino acid sequence shown in SEQ ID NO:10. In some embodiments, the first constant region comprises the amino acid sequence shown in SEQ ID NO:3, and the second constant region comprises the amino acid sequence shown in SEQ ID NO:13. In some preferred embodiments, the first constant region comprises the amino acid sequence shown in SEQ ID NO:6, and the second constant region comprises the amino acid sequence shown in SEQ ID NO:13.

[0081] In some embodiments, the α-chain and / or β-chain, preferably the α-chain and β-chain, have at least one exogenous intracellular functional domain attached to their C-terminus. In some embodiments, the exogenous intracellular functional domain is connected directly or via a linker to the α-chain and / or β-chain, preferably to the C-terminus of the constant region of the α-chain and β-chain. In some embodiments, the exogenous intracellular functional domain is connected via a linker to the α-chain and / or β-chain missing from the intracellular region, preferably to the C-terminus of the constant region of the α-chain and β-chain. In some embodiments, the linker is a (G4S)n linker, where n represents an integer from 1 to 10, preferably n is 3.

[0082] In some embodiments, the first constant region is a modified TCRα chain constant region derived from the mouse TCRα chain constant region, wherein, relative to the wild-type mouse TCRα chain constant region, the amino acid at position 48, such as threonine (T), is mutated to cysteine ​​(C), the amino acid at position 112, such as serine (S), is replaced with leucine (L), the amino acid at position 114, such as methionine (M), is replaced with isoleucine (I), and the amino acid at position 115, such as glycine (G), is replaced with valine (V), and the α chain comprises an intracellular domain of OX40 connected to the C-terminus of the constant region (e.g., via a linker, such as a (G4S)n linker, where n represents an integer from 1 to 10, preferably n is 3); and

[0083] The second constant region is a modified TCRβ chain constant region derived from the mouse TCRβ chain constant region, wherein, relative to the wild-type mouse TCRβ chain constant region, the amino acid at position 56, for example serine S, is mutated to cysteine ​​C, and the β chain contains an intracellular domain of OX40 connected to the C-terminus of the constant region (e.g., via a linker, such as (G4S)n linker, where n represents an integer from 1 to 10, preferably n is 3).

[0084] As used in this article, “exogenous” means a protein or nucleic acid sequence that is derived from a foreign species, or, if derived from the same species, a protein or nucleic acid sequence whose composition and / or location have been significantly altered from its natural form through deliberate human intervention.

[0085] As used in this article, "exogenous intracellular functional domain" can be the intracellular domain of co-stimulatory molecules such as CD40, OX40, ICOS, CD28, 4-1BB, CD27, and CD137; it can also be the intracellular domain of co-inhibitory molecules, such as TIM3, PD1, CTLA4, and LAG3; it can also be the intracellular domain of cytokine receptors such as interleukin receptors (e.g., IL-2β, IL-7α, or IL-21 receptors), interferon receptors, tumor necrosis factor superfamily receptors, colony-stimulating factor receptors, chemokine receptors, growth factor receptors, or other membrane proteins; or the domain of intracellular proteins such as NIK.

[0086] In some preferred embodiments, the exogenous intracellular functional domain is an intracellular domain of a co-stimulatory molecule, preferably an intracellular domain of OX40. In some embodiments, the intracellular domain of OX40 comprises the amino acid sequence of SEQ ID NO:15.

[0087] In some implementations, the antigen-binding region is fused directly or indirectly (e.g., via a linker) to the N-terminus of the first and / or second constant region.

[0088] In some embodiments, the α chain includes a first antigen-binding region and a first constant region, and the β chain includes a second constant region. In this case, the β chain does not contain an antigen-binding region.

[0089] In some embodiments, the α chain includes a first constant region, and the β chain includes a second antigen-binding region and a second constant region. In this case, the α chain does not contain an antigen-binding region.

[0090] In some embodiments, the α chain includes a first antigen-binding region and a first constant region, and the β chain includes a second antigen-binding region and a second constant region.

[0091] In some embodiments, the first antigen-binding region and the second antigen-binding region each bind specifically to the target antigen independently or in combination. Those skilled in the art will understand that when the antigen-binding region contains a single-domain antibody or a single-chain antibody, it can bind to the target antigen alone. However, if the first antigen-binding region contains a conventional antibody heavy chain variable region and the second antigen-binding region contains a conventional antibody light chain variable region, then the first and second antigen-binding regions bind to the target antigen in combination, and vice versa.

[0092] The target antigen described in this invention can be a disease-related antigen, preferably a cancer-related antigen, such as those selected from the following cancer-related antigens: GPC3 (phosphatidylinositol proteoglycan 3), BCMA, mesothelin, LILRB4, CD16, CD64, CD78, CD96, CLL1, CD7, CD70, CD38, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2 / neu), Claudin 18.2, and carcinoembryonic antigen (CEA). Epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD276, CD19, CD22, CD20, CD30, CD40, disialotetrahexosylganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alpha fetal globulin (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostaglandin, prostaglandin-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, Prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, liver glycoside B2, CD22, insulin-like growth factor (IGF1)-I, IGF-II, IGFI receptor, major histocompatibility complex (MHC) molecule presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor matrix antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, A1 domain (TnC) of tendin-C A1), fibroblast-associated protein (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, Foxp3, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endothelial factors, major histocompatibility complex (MHC) molecules, TNFRSF17, SLAMF7, GPRC5D, FKBP11, KAMP3, ITGA8, and FCRL5.

[0093] In some embodiments, the target antigen is an antigen derived from a pathogen or a surface antigen of cells infected by a pathogen, such as RSVF (prevention of respiratory syncytial virus), PA (inhalation anthrax), CD4 (HIV infection), etc.

[0094] In some implementations, the target antigen is a molecule produced or secreted by disease-causing cells, such as CD3 (involved in transplant rejection), CD25 (involved in acute kidney transplant rejection), C5 (involved in paroxysmal nocturnal hemoglobinuria), IL-1β (involved in cold pyridine-associated periodic syndrome), RANKL (involved in cancer-related bone injury), von Willebrand factor (involved in adult acquired thrombotic thrombocytopenic purpura), plasma kallikrein (involved in angioedema), calcitonin gene-related peptide receptor (involved in adult migraine), FGF23 (involved in X-linked hypophosphatemia), etc.

[0095] The antigen-binding region may be derived from one or more known antibodies, including any commercially available antibody such as FMC63, rituximab, alemtuzumab, epratuzumab, trastuzumab, bivatuzumab, cetuximab, labetuzumab, palivizumab, sevirumab, and tuvirumab. Baliximab, daclizumab, infliximab, omalizumab, efalizumab, keliximab, siplizumab, natalizumab, clenoliximab, pemtumomab, edrecolomab, cantuzumab, etc.

[0096] In some embodiments, the first antigen-binding region comprises a heavy chain variable region of an antibody that specifically binds to the target antigen, and the second antigen-binding region comprises a light chain variable region of the antibody; or, the first antigen-binding region comprises a light chain variable region of an antibody that specifically binds to the target antigen, and the second antigen-binding region comprises a heavy chain variable region of the antibody.

[0097] In some embodiments, the first antigen-binding region contains a single-chain antibody (e.g., scFv) or a single-domain antibody that specifically binds to the target antigen; and / or the second antigen-binding region contains a single-chain antibody or a single-domain antibody that specifically binds to the target antigen.

[0098] In some embodiments, the single-chain antibody (e.g., scFv) comprises a heavy chain variable region and a light chain variable region connected by a linker, such as a flexible linker. Those skilled in the art can readily utilize existing knowledge and methods to construct single-chain antibodies from the heavy chain variable regions and light chain variable regions of conventional antibodies.

[0099] In some implementations, the first antigen-binding region and the second antigen-binding region bind the same target antigen.

[0100] In some implementations, the first antigen-binding region and the second antigen-binding region bind to different regions (e.g., different epitopes) of the same target antigen.

[0101] In some implementations, the first antigen-binding region and the second antigen-binding region bind different target antigens.

[0102] In some embodiments, the antigen-binding region comprises a heavy chain variable region and / or a light chain variable region of an antibody that specifically binds to GPC3, wherein the heavy chain variable region comprises VH CDR1 of SEQ ID NO:73 (GYTFTDYE), VH CDR2 of SEQ ID NO:74 (LDPKTGDT), and VH CDR3 of SEQ ID NO:75 (TR); and the light chain variable region comprises VL CDR1 of SEQ ID NO:76 (QSLVHSNRNTY), VL CDR2 of SEQ ID NO:77 (KVS), and VL CDR3 of SEQ ID NO:78 (SQNTHVP).

[0103] In some embodiments, the antigen-binding region comprises the heavy chain variable region shown in SEQ ID NO:22 and / or the light chain variable region shown in SEQ ID NO:23, thereby the STAR targets GPC3. In some embodiments, the first antigen-binding region comprises the heavy chain variable region shown in SEQ ID NO:22, and the second antigen-binding region comprises the light chain variable region shown in SEQ ID NO:23. In some embodiments, the first antigen-binding region comprises the light chain variable region shown in SEQ ID NO:23, and the second antigen-binding region comprises the heavy chain variable region shown in SEQ ID NO:22. In some embodiments, the first antigen-binding region comprises the heavy chain variable region shown in SEQ ID NO:22 and the light chain variable region shown in SEQ ID NO:23 (e.g., comprising the single-chain antibody sequence shown in SEQ ID NO:24). In some preferred embodiments, the second antigen-binding region comprises the heavy chain variable region shown in SEQ ID NO:22 and the light chain variable region shown in SEQ ID NO:23 (e.g., comprising the single-chain antibody sequence shown in SEQ ID NO:24).

[0104] In some embodiments, the antigen-binding region comprises a single-domain antibody sequence that specifically binds mesothelin (MSLN), comprising CDR1 of SEQ ID NO:61, CDR2 of SEQ ID NO:62, and CDR3 of SEQ ID NO:63; or comprising CDR1 of SEQ ID NO:64, CDR2 of SEQ ID NO:65, and CDR3 of SEQ ID NO:66.

[0105] In some embodiments, the antigen-binding region comprises a single-domain antibody sequence selected from SEQ ID NO:25 or 26, thereby enabling the STAR to target mesothelin (MSLN). In some embodiments, the first antigen-binding region or the second antigen-binding region, preferably the second antigen-binding region, comprises a single-domain antibody sequence selected from SEQ ID NO:25 or 26. In some embodiments, the first antigen-binding region comprises a single-domain antibody sequence of SEQ ID NO:25, and the second antigen-binding region comprises a single-domain antibody sequence of SEQ ID NO:26. In some embodiments, the first antigen-binding region comprises a single-domain antibody sequence of SEQ ID NO:26, and the second antigen-binding region comprises a single-domain antibody sequence of SEQ ID NO:25.

[0106] In some embodiments, the antigen-binding region comprises a single-domain antibody sequence that specifically binds to LILRB4, comprising CDR1 of SEQ ID NO:67, CDR2 of SEQ ID NO:68, and CDR3 of SEQ ID NO:69; or comprising CDR1 of SEQ ID NO:70, CDR2 of SEQ ID NO:71, and CDR3 of SEQ ID NO:72.

[0107] In some embodiments, the antigen-binding region comprises a single-domain antibody sequence selected from SEQ ID NO:27 or 28, thereby enabling the STAR to target LILRB4. In some embodiments, the first antigen-binding region or the second antigen-binding region, preferably the second antigen-binding region, comprises a single-domain antibody sequence selected from SEQ ID NO:27 or 28. In some embodiments, the first antigen-binding region comprises a single-domain antibody sequence of SEQ ID NO:27, and the second antigen-binding region comprises a single-domain antibody sequence of SEQ ID NO:28. In some embodiments, the first antigen-binding region comprises a single-domain antibody sequence of SEQ ID NO:28, and the second antigen-binding region comprises a single-domain antibody sequence of SEQ ID NO:27.

[0108] In some embodiments, the antigen-binding region comprises a single-domain antibody sequence that specifically binds to BCMA, comprising CDR1 of SEQ ID NO:46, CDR2 of SEQ ID NO:47, and CDR3 of SEQ ID NO:48; or comprising CDR1 of SEQ ID NO:49, CDR2 of SEQ ID NO:50, and CDR3 of SEQ ID NO:51; or comprising CDR1 of SEQ ID NO:52, CDR2 of SEQ ID NO:53, and CDR3 of SEQ ID NO:54; or comprising CDR1 of SEQ ID NO:55, CDR2 of SEQ ID NO:56, and CDR3 of SEQ ID NO:57; or comprising CDR1 of SEQ ID NO:58, CDR2 of SEQ ID NO:59, and CDR3 of SEQ ID NO:60.

[0109] In some embodiments, the antigen-binding region comprises a single-domain antibody sequence selected from SEQ ID NO:29-33, thereby enabling the STAR to target BCMA. In some embodiments, either the first antigen-binding region or the second antigen-binding region, preferably the second antigen-binding region, comprises a single-domain antibody sequence selected from SEQ ID NO:29-33. In some embodiments, the first antigen-binding region comprises one single-domain antibody sequence selected from SEQ ID NO:29-33, and the second antigen-binding region comprises another single-domain antibody sequence selected from SEQ ID NO:29-33.

[0110] In some embodiments, the first antigen-binding region comprises a single-domain antibody sequence selected from SEQ ID NO:27 or 28, and the second antigen-binding region comprises a single-domain antibody sequence selected from SEQ ID NO:29-33, thereby the STAR targets LILRB4 and BCMA. In some embodiments, the first antigen-binding region comprises a single-domain antibody sequence selected from SEQ ID NO:29-33, and the second antigen-binding region comprises a single-domain antibody sequence selected from SEQ ID NO:27 or 28, thereby the STAR targets LILRB4 and BCMA. In some preferred embodiments, the first antigen-binding region comprises a single-domain antibody sequence of SEQ ID NO:28, and the second antigen-binding region comprises a single-domain antibody sequence of SEQ ID NO:29, thereby the STAR targets LILRB4 and BCMA.

[0111] In some embodiments, the α chain comprises the amino acid sequence shown in SEQ ID NO:39, and the β chain comprises the amino acid sequence shown in SEQ ID NO:38. (Corresponding to GC33-STAR)

[0112] In some embodiments, the α chain comprises the amino acid sequence shown in SEQ ID NO:41, and the β chain comprises the amino acid sequence shown in SEQ ID NO:40 (corresponding to NM5-STAR).

[0113] In some embodiments, the α chain comprises the amino acid sequence shown in SEQ ID NO:43, and the β chain comprises the amino acid sequence shown in SEQ ID NO:42 (corresponding to NLB4-NLB14-STAR).

[0114] In some embodiments, the α chain comprises the amino acid sequence shown in SEQ ID NO:45, and the β chain comprises the amino acid sequence shown in SEQ ID NO:44 (corresponding to NBC11-NLB14-STAR).

[0115] In some embodiments, the α chain comprises the amino acid sequence shown in SEQ ID NO:83, and the β chain comprises the amino acid sequence shown in SEQ ID NO:84. (Corresponding to NBC11-STAR)

[0116] The immune cells described in this invention include, but are not limited to, T cells or NK cells, preferably T cells.

[0117] The immune cells, such as T cells, of the present invention can be obtained from a variety of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, ascites, pleural effusion, spleen tissue, and tumors, by various non-limiting methods. In some embodiments, the cells can be derived from healthy donors or from patients diagnosed with cancer. In some embodiments, the cells can be part of a mixed population of cells exhibiting different phenotypic characteristics. For example, immune cells such as T cells can be obtained by isolating peripheral blood mononuclear cells (PBMCs) and then activating and expanding them with specific antibodies.

[0118] In some embodiments, the immune cells, such as T cells, described in this invention are isolated (ex vivo) immune cells, such as cells.

[0119] In some embodiments of various aspects of the invention, the immune cells, such as T cells, are derived from the subject's own cells. As used herein, "autologous" means that the cells, cell lines, or cell populations used to treat the subject are derived from the subject. In some embodiments, the immune cells, such as T cells, are derived from allogeneic cells, for example, from a donor compatible with the subject's human leukocyte antigen (HLA). Cells from the donor can be converted into non-allogeneic reactive cells using standard protocols and replicated as needed to produce cells that can be administered to one or more patients.

[0120] Expression vectors and methods for preparing therapeutic immune cells

[0121] In one aspect, the present invention provides an expression vector comprising the coding sequence of a synthetic T-cell receptor antigen receptor (STAR) as defined above and the coding sequence of a membrane-bound IL-15 protein (mbIL-15) as defined above.

[0122] The coding sequence in the expression vector of the present invention can be operatively linked to a regulatory element such as a promoter for expression in cells. Examples of promoters include the MND promoter or the HTLV promoter. An exemplary nucleotide sequence of the MND promoter is shown in SEQ ID NO:34; the MND promoter may contain a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or even at least 99% sequence identity with SEQ ID NO:34. An exemplary nucleotide sequence of the HTLV promoter is shown in SEQ ID NO:35; the HTLV promoter may contain a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or even at least 99% sequence identity with SEQ ID NO:35. Preferably, the promoter is an MND promoter.

[0123] In some embodiments, the expression vector comprises

[0124] a) A coding nucleotide sequence comprising a fusion polypeptide of the α chain of the STAR, the β chain of the STAR, and the mbIL-15 linked by a self-cleaving peptide, preferably, the coding nucleotide sequence being operatively linked to an MND promoter; or

[0125] b) comprising the encoding nucleotide sequence of a fusion polypeptide of the α and β chains of the STAR linked by a self-cleaving peptide, and the encoding nucleotide sequence of mbIL-15, wherein the encoding nucleotide sequence of the α and β chain fusion polypeptide and the encoding nucleotide sequence of mbIL-15 are each independently operatively linked to a promoter, or the encoding nucleotide sequence of the α and β chain fusion polypeptide and the encoding nucleotide sequence of mbIL-15 are linked via an internal ribosome entry site (IRES), thereby achieving co-expression of the STAR and the mbIL-15.

[0126] As used herein, "self-cleaving peptide" refers to a peptide capable of self-cleaving within a cell. For example, the self-cleaving peptide may contain a protease recognition site, thereby being recognized and specifically cleaved by intracellular proteases. Alternatively, the self-cleaving peptide may be a 2A peptide. 2A peptides are a class of short peptides derived from viruses whose self-cleavage occurs during translation. When two different target proteins are expressed in the same reading frame using a 2A peptide, the two target proteins are generated in an almost 1:1 ratio. Commonly used 2A peptides include P2A from porcine techovirus-1, T2A from the β-tetrasomatic moth virus (Thosea asigna virus), E2A from equine rhinitis A virus, and F2A from foot-and-mouth disease virus. P2A has the highest cleavage efficiency and is therefore preferred. Various functional variants of these 2A peptides are also known in the art and can also be used in this invention. 2A peptides can also be combined with a Furin recognition sequence to remove additional introduced amino acid sequences.

[0127] In some embodiments, the self-cleaving peptide is a 2A peptide, such as a P2A peptide. In some embodiments, the self-cleaving peptide is a Furin-2A peptide, such as the Furin-P2A peptide shown in SEQ ID NO:17.

[0128] In some embodiments, the different portions of the fusion polypeptide can be arranged in different ways, as long as they are separated by self-cleaving peptides. For example, in some embodiments, the fusion polypeptide may include the β chain, a self-cleaving peptide such as Furin-P2A, and the α chain from the N-terminus to the C-terminus. In some embodiments, the fusion polypeptide may include the β chain, a self-cleaving peptide such as Furin-P2A, the α chain, a self-cleaving peptide such as Furin-P2A, and the mbIL-15 from the N-terminus to the C-terminus. When multiple self-cleaving peptides are present, the self-cleaving peptides may be the same or different.

[0129] In some embodiments, the α chain, the β chain, and / or the mbIL-15 encoded by the expression vector further include an N-terminal signal peptide. In some embodiments, the α chain and / or the β chain include a GM-CSF signal peptide (SEQ ID NO:37) at its N-terminus. In some embodiments, the mbIL-15 includes an IgE signal peptide at its N-terminus, such as the IgE signal peptide shown in SEQ ID NO:36.

[0130] In another aspect, the present invention provides a method for preparing therapeutic immune cells, comprising:

[0131] Step 1) Provide initial immune cells;

[0132] Step 2) Introduce the expression vector of the present invention into the initiating immune cells; and

[0133] Step 3) Harvest the immune cells obtained in Step 2).

[0134] In some embodiments, the initiating immune cell is a T cell. In other embodiments, the initiating immune cell is an NK cell.

[0135] The initiating immune cells, such as T cells, of the present invention can be obtained from a variety of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, ascites, pleural effusion, spleen tissue, and tumors, using various non-limiting methods. In some embodiments, the cells can be derived from healthy donors or from patients diagnosed with cancer. In some embodiments, the cells can be part of a mixed population of cells exhibiting different phenotypic characteristics. For example, initiating immune cells, such as T cells, can be obtained by isolating peripheral blood mononuclear cells (PBMCs) and then activating and expanding them with specific antibodies.

[0136] In some embodiments, the initiating immune cells, such as T cells, described in this invention are isolated (ex vivo) immune cells, such as T cells. Therefore, the therapeutic immune cells, such as T cells, obtained by this invention are isolated (ex vivo) therapeutic immune cells, such as T cells.

[0137] In some embodiments, the method described in this invention is an in vitro method.

[0138] In some embodiments of various aspects of the invention, the initiating immune cells, such as T cells, are derived from the subject's own cells. As used herein, "autologous" means that the cells, cell lines, or cell populations used to treat the subject are derived from the subject. In some embodiments, the initiating immune cells, such as T cells, are derived from allogeneic cells, for example, from a donor compatible with the subject's human leukocyte antigen (HLA). Cells from the donor can be converted into non-allogeneic reactive cells using standard protocols and replicated as needed to produce cells that can be administered to one or more patients.

[0139] The expression vector can be introduced into immune cells such as T cells by methods known in the art, including but not limited to microinjection, electroporation, virus-mediated transfection, and liposome-mediated transfection.

[0140] In some embodiments, the method further includes, between steps 2) and 3), step x) amplifying immune cells, such as T cells, obtained in step 2). Immune cells, such as T cells, can be amplified using methods known in the art.

[0141] In some embodiments, the method further includes step y) screening for immune cells, such as T cells, that co-express mbIL-15 and the STAR. In some embodiments, step y) may be performed after step 2). In some embodiments, step y) may be performed after step 2) and before step x). In some embodiments, step y) may be performed after step x). In some embodiments, the screening is performed by flow cytometry.

[0142] In another aspect, the present invention provides therapeutic immune cells such as T cells comprising the expression vector of the present invention, or therapeutic immune cells such as T cells that can be obtained or acquired by the expression vector of the present invention or the method of the present invention.

[0143] Pharmaceutical Compositions and Applications

[0144] In another aspect, the present invention provides a pharmaceutical composition comprising the therapeutic immune cells of the present invention and / or the expression vector of the present invention, and a pharmaceutically acceptable carrier.

[0145] In another aspect, the present invention provides the use of the therapeutic immune cells of the present invention, the expression vectors of the present invention, and / or the pharmaceutical compositions of the present invention in the preparation of medicaments for treating diseases in subjects.

[0146] In another aspect, the present invention provides a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the therapeutic immune cells of the present invention, the expression vector of the present invention, and / or the pharmaceutical composition of the present invention.

[0147] In practical applications, the dosage levels of cells and / or expression vectors in the pharmaceutical compositions of this invention may be varied to obtain an amount of active ingredient that effectively achieves the desired therapeutic response for a specific patient, composition, and route of administration, while being non-toxic to the patient. The selected dosage level depends on a variety of pharmacokinetic factors, including the activity of the specific composition of this invention applied, the route of administration, the time of administration, the excretion rate of the specific compound applied, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition applied, the age, sex, weight, condition, general health status, and medical history of the patient receiving treatment, and similar factors known in the medical field.

[0148] The expression vector, therapeutic immune cell, or pharmaceutical composition or drug according to the present invention can be administered in any convenient manner, including by injection, infusion, implantation, or transplantation. The expression vector, therapeutic immune cell, or pharmaceutical composition described herein can be administered intravenously, intralymphaticly, intradermally, intratumorally, intramedullaryly, intramuscularly, or intraperitoneally. In one embodiment, the expression vector, therapeutic immune cell, or pharmaceutical composition of the present invention is preferably administered by intravenous injection.

[0149] In embodiments of various aspects of the present invention, the disease depends on the target antigen targeted by the STAR.

[0150] In embodiments of various aspects of the present invention, the disease is, for example, cancer, and examples of such cancers include, but are not limited to, myeloma, lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, laryngeal cancer, cervical cancer, endometrial tumors, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancers, thyroid cancer, parathyroid cancer, etc. Adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and combinations of the aforementioned cancers.

[0151] In various embodiments of the present invention, the disease is, for example, a pathogen infection, and examples of the pathogens include, but are not limited to, respiratory syncytial virus, anthrax, and human immunodeficiency virus.

[0152] In various embodiments of the present invention, the disease is, for example, cardiovascular disease, diabetes, neurological disease, post-transplant rejection, or some other disease.

[0153] In various embodiments of the present invention, the disease may also be an autoimmune disease, including but not limited to systemic lupus erythematosus (SLE), myositis, scleroderma, Sjögren's syndrome, autoimmune hemolytic anemia, and rheumatoid arthritis.

[0154] In embodiments of various aspects of the present invention, the disease is a phosphatidylinositol proteoglycan-3 (GPC3)-related disease, such as a disease related to abnormal GPC3 expression, or GPC3-related cancer. The cancer is, for example, liver cancer such as hepatocellular carcinoma, lung cancer such as squamous cell carcinoma (SqCC), gastric cancer, ovarian cancer, melanoma, or pediatric embryonal tumors.

[0155] In embodiments of various aspects of this invention, the disease is a B-cell maturation antigen (BCMA)-related disease, such as a disease related to abnormal BCMA expression, such as BCMA-related cancer. The cancer is, for example, myeloma, such as multiple myeloma (MM), particularly relapsed or refractory multiple myeloma (RRMM); or an autoimmune disease, including but not limited to systemic lupus erythematosus (SLE), myositis, scleroderma, Sjögren's syndrome, autoimmune hemolytic anemia, and rheumatoid arthritis.

[0156] In embodiments of various aspects of the present invention, the disease is a mesothelin (MSLN)-related disease, such as a disease related to abnormal MSLN expression, such as MSLN-related cancer. Examples of such cancers include ovarian cancer, breast cancer, colorectal cancer, and pancreatic cancer.

[0157] In embodiments of various aspects of the present invention, the disease is a leukocyte immunoglobulin-like receptor B4 (LILRB4) related disease, such as a disease related to abnormal LILRB4 expression, such as LILRB4-related cancer. The cancer is, for example, leukemia, particularly acute myeloid leukemia.

[0158] Example

[0159] Example 1: STAR Optimization

[0160] B cell secretory antibodies (Abs) or B cell receptors (BCRs) share significant similarities with T cell receptors (TCRs) in gene structure, protein structure, and spatial conformation. Both antibodies and TCRs consist of variable and constant regions. The variable region is responsible for antigen recognition and binding, while the constant region plays a role in structural interaction and signal transduction. By replacing the variable regions of the TCRα and β chains (or TCRγ and δ chains) with the heavy chain variable regions (VH) and light chain variable regions (VL) of the antibody, a synthetic chimeric molecule called a synthetic T-cell receptor and antibody receptor (STAR) can be constructed.

[0161] The STAR molecule has two chains. The first chain is formed by fusing an antigen recognition sequence (such as the variable region VH of the antibody heavy chain) with the constant region (Cα) of the T cell receptor α chain (TCRα). The second chain is formed by fusing an antigen recognition sequence (such as the variable region VL of the antibody light chain) with the constant region (Cβ) of the T cell receptor β chain (TCRβ). The antigen recognition domains (such as VH, VL, or scFv) and constant region domains (the constant regions of TCRα, β, γ, and δ) in this construct can be arranged and combined to form various constructs with different configurations but similar functions.

[0162] After the first and second chains of the STAR molecule are expressed in T cells, they bind with endogenous CD3εδ, CD3γε, and CD3ζζ chains in the endoplasmic reticulum to form an eight-subunit complex, which is then displayed on the cell membrane surface as a complex. The immunoreceptor tyrosine-based activation motif (ITAM) is a signal transduction motif in the TCR molecule, with a conserved sequence of YxxL / V. The intracellular regions of the CD3ε, δ, γ, and ε chains contain one ITAM sequence, and the intracellular region of the CD3ζ chain contains three ITAM sequences, so a complete STAR complex contains a total of 10 ITAM sequences. When the antigen recognition sequence of the STAR receptor binds to its specific antigen, the intracellular ITAM sequences are successively phosphorylated, thereby activating downstream signaling pathways, activating transcription factors such as NF-κB, NFAT, and AP-1, triggering T cell activation, and producing effector functions.

[0163] The inventors have previously modified the constant region of STAR to improve its performance. Specifically, this includes:

[0164] First, the constant region is modified to be mouse-derived: Since the constant region sequences of human, primate and mouse TCRα / β chains (mouse TCRAC / mouse TCRBC) are highly conserved in function and have the same key amino acid sequences, they can be substituted for each other. After substitution, the efficiency of correct pairing of STAR molecules is increased, the possibility of mismatches causing unknown specificity is reduced and the safety is increased.

[0165] Secondly, point mutations were introduced into disulfide bonds using cysteine: a threonine T mutation at position 48 was replaced with a cysteine ​​C mutation in the constant region of the murine TCR α chain, and a serine S mutation at position 56 was replaced with a cysteine ​​C mutation in the constant region of the murine TCR β chain. These two newly added cysteine ​​residues form disulfide bonds between the two STAR chains, reducing mismatches between the two STAR chains and the endogenous TCR chain, and helping the STAR molecule form a more stable complex. The obtained α chain constant region was named TRAC(Cys), and the obtained β chain constant region was named TRBC(Cys).

[0166] The STAR transmembrane region was designed with hydrophobic amino acid substitutions: Mutations were made at three amino acid sites within the transmembrane region of the TCR α-chain constant region, from amino acid positions 111 to 119. Serine (S) at position 112 was replaced with leucine (L), methionine (M) at position 114 with isoleucine (I), and glycine (G) at position 115 with valine (V). The overall amino acid sequence of this region changed from LSVGMLRIL to LLVIVLRIL. This design increased the hydrophobicity of the transmembrane region, counteracting the instability caused by the positive charge carried by the TCR transmembrane region, allowing the STAR molecule to exist more stably on the cell membrane and thus acquire better function. The α-chain constant region obtained by combining cysteine ​​and hydrophobic region mutations was named TRAC(Cys-TM), and the corresponding β-chain constant region was named TRBC(Cys-TM), where TRBC(Cys-TM) is identical to TRBC(Cys).

[0167] To further optimize the design of the STAR molecule, a specific rearrangement was performed on the N-terminus of the STAR molecule's constant region, based on murine derivatization of the constant region, cysteine ​​point mutations, and hydrophobic amino acid mutations in the α-chain constant region, to achieve better results. Rearrangement involves partial sequence deletion and humanization mutation of other sequences. The significance of humanization mutation lies in minimizing non-human sequences in the STAR molecule while maintaining its function, thereby minimizing the possibility of receptor rejection of STAR-T cells in clinical applications. Therefore, the N-terminus of the TCR α-chain constant region was further modified, including replacing amino acid E at position 6 with D, replacing K at position 13 with R, and deleting amino acids 15-18. The resulting α-chain constant region was named TRAC (Nrec-Cys-TM). Further modifications were made to the N-terminal 25 amino acids of the TCR β-chain constant region, including replacing the 3rd amino acid (R) with K, the 6th amino acid (T) with F, the 9th amino acid (K) with E, the 11th amino acid (S) with A, and the 12th amino acid (L) with V, and the 17th and 21-25th amino acids were deleted. The resulting β-chain constant region was named TRBC (Nrec-Cys-TM).

[0168] Furthermore, STAR function can be further enhanced by linking co-stimulatory molecules, such as the OX40 cytoplasmic region, to the C-terminus of the α-chain constant region and / or the β-chain constant region. The co-stimulatory molecules can be directly linked or linked via a linker, such as a (G4S)3 linker, to the C-terminus of the α-chain constant region and / or the β-chain constant region. In addition to the modifications described above, the constant regions linked to the co-stimulatory molecules can also lack the native intracellular region relative to the wild-type constant region, which further improves STAR function. For example, the α-chain constant region may lack amino acids 136-137; and / or, the β-chain constant region may lack amino acids 167-172.

[0169] Example 2: Screening for proteins co-expressed with STAR

[0170] 2.1. MSLN STAR-T structural design for co-expression of different proteins

[0171] Previously, the inventors obtained the MSLN-specific nanobody NM5 (SEQ ID NO:25) using a nanobody screening platform. Membrane protein array chip technology demonstrated that the NM5 antibody specifically binds to MSLN, indicating good safety. BLI affinity assays and competition experiments revealed that NM5 has a high affinity for MSLN (KD = 1.14E-08M) and a unique juxtamembranous recognition epitope. Based on this, NM5STAR was constructed. The α-chain constant region of NM5 STAR is based on TRAC (Cys-TM), and the β-chain constant region is based on TRBC (Cys-TM). Both the α-chain and β-chain constant regions are directly linked to the OX40 cytoplasmic region. The NM5 nanobody is fused to the N-terminus of the β-chain constant region. The α-chain amino acid sequence of NM5 STAR is shown in SEQ ID NO:41; the β-chain amino acid sequence is shown in SEQ ID NO:40. The α and β chains of NM5 STAR also contain the GM-CSF signal peptide (SEQ ID NO:37) at the N-terminus, which is cleaved in the cell after expression.

[0172] Compared to other nanobodies, T cells expressing NM5 STAR exhibit stronger in vitro and in vivo antitumor effects. Building upon this research, MSLN STAR-T cells were modified to enhance their persistence and, consequently, their in vivo antitumor activity. mbIL-15, sIL-15, p52, or IL-10 were linked to MSLN STAR cells via furin-P2A (Figure 1). The mbIL-15 (SEQ ID NO:21) design involved linking IL-15 (SEQ ID NO:18) and IL-15Rα (SEQ ID NO:19) via a flexible linker (SEQ ID NO:20), with an IgE signal peptide (SEQ ID NO:36) added to the N-terminus. Fusion protein expression was driven by the HTLV promoter (SEQ ID NO:35).

[0173] 2.2. Vector construction and viral packaging for co-expressing different proteins in MSLN STAR

[0174] Commercially synthesized and assembled coding nucleic acid sequences with different structures as shown in Figure 1 were obtained. The MSLN nanobody NM5 sequence was assembled into the constant regions (TRAC(Cys-TM) and TRBC(Cys-TM)) of the STAR molecule. The STAR contains an OX40 co-stimulatory domain that is linked to the C-terminus of the TRAC or TRBC constant region via a linker or directly. Homologous recombination was used to insert the sequences into lentiviral expression vectors to construct plasmids with different MSLN STAR structures for co-expressing different proteins.

[0175] Lentix-293T cells were divided into groups of 5 × 10 5 Inoculate cells / mL into 10cm culture dishes and transfect when the cell density reaches about 80%. The ratio of the four plasmids is PMD2.G:PRSV-Rev:PMDlg:transfer plamid = 1:1:2:4. The volume-to-mass ratio of PEI-Max to plasmid is 3:1. Change the medium after 12-16 hours and collect the virus solution after 48 hours and 72 hours.

[0176] The virus was serially diluted 10-fold in 96-well plates, and then Jurkat-C5 cells with TCR knockout were incubated at 1.5 × 10⁻⁶ wells. 5 Virus cells / mL were added to the wells, centrifuged at 32°C and 1500 rpm for 90 min, and incubated in an incubator. After 72 h, the infection efficiency was measured by flow cytometry. Wells with an infection rate of 2-30% were selected for titer calculation. Titrate (TU / mL) = 1.5 × 10⁻⁶. 4 × Positive rate ÷ Viral volume (μL) × 1000.

[0177] 2.3. In vitro functional assay of MSLN-STAR-T cells co-expressing different proteins

[0178] 1) Isolation, activation, and infection of human primary T cells

[0179] PBMCs were isolated using the Ficoll method, and cell counts were performed at 1 × 10⁶ cells per cell. 6 Cells were activated by adding 10 μl of CD3 / CD28TransAct at a concentration of 1.2 × 10⁻⁶. 6 The cells were cultured at a density of 1 / mL, and after 24 hours, they were infected with the virus at an MOI of 2. After 24 hours, the medium was changed, and the cells were passaged every other day.

[0180] 2) STAR infection efficiency test

[0181] 72 hours after infection, the STAR infection efficiency was analyzed by measuring the mTCRβ ratio using flow cytometry (Figure 2).

[0182] 3) Protein co-expression verification

[0183] MSLN STAR-T cells expressing different proteins, along with their culture supernatants and proteins, were collected. Flow cytometry, ELISA, and Western blotting were used to detect mbIL-15 on the cell membrane surface (at different MOI values ​​of 4 / 8 / 16), sIL-15 and IL-10 in the supernatant, and p52 expression at the protein level. The results indicated that all four proteins could be successfully expressed (Figures 3A-3D).

[0184] 4) Killing efficiency of MSLN STAR-T cells co-expressing different proteins against different target cells

[0185] Aspc-1 (human metastatic pancreatic adenocarcinoma cells) and SKOV3 (human ovarian cancer cells) target cells were 1.5 × 10⁻⁶ cells per cell line. 5 The cells were seeded at a density of 0.3:1, 0.1:1, and 1:1 with STAR-T cells in 24-well plates. After 24 hours, STAR-T cells were added to the target cells at ratios of 0.3:1, 0.1:1, and 1:1. After 24 hours of culture, the killing efficiency of STAR-T cells against the target cells was detected (Figures 4A-4B). As shown in the figures, when the SKOV3 target cell effector-to-target ratio was 0.3:1, the killing level of IL-10 was the best, followed by P52, then mbIL-15 and sIL-15; when the effector-to-target ratio was 0.1:1, the killing level of IL-10 was the best, followed by P52 and mbIL-15 with comparable killing effects, then sIL-15. For Aspc-1 target cells, when the effector-to-target ratio is 0.3:1, the killing levels of IL-10 and mbIL-15 are equivalent, followed by P52 and sIL-15. When the effector-to-target ratio is 1:1, the killing levels of IL-10 and mbIL-15 are equivalent, followed by P52, and lastly sIL-15.

[0186] 2.4. In vivo efficacy evaluation of MSLN STAR-T co-expressing different proteins

[0187] The effects of different modifications on the in vivo efficacy and proliferation of MSLN STAR-T cells were investigated using an Aspc-1 subcutaneous tumor model. Fluorescently labeled Aspc-1 cells were subcutaneously injected into NCG-immunodeficient mice to construct an Aspc-1 cell-mediated NCG mouse tumor in vivo imaging model. On day 4 after Aspc-1 cell infusion, 7 × 10T MSLN STAR-T cells co-expressing different proteins were infused via tail vein. 6(each cell) was then examined, and tumor growth was monitored every 3-4 days using an in vivo imaging system. The results (Figures 5A-5C) showed that mbIL-15 and sIL-15 modified MSLN STAR-T cells exhibited significantly enhanced anti-tumor effects in vivo, while simultaneously exacerbating GVHD responses. However, mbIL-15 demonstrated superior safety and efficacy compared to sIL-15. Analysis of T cell proliferation in vivo revealed a significant increase in the number of T cells and STAR+ T cells in the mbIL-15STAR and sIL-15STAR groups, approximately 10 times that of the control group. Furthermore, mbIL-15STAR and sIL-15STAR maintained a higher proportion of STAR-positive and CD8-positive cells, significantly improving the survival and persistence of STAR-T and CD8+ T cells (Figures 6A-6D).

[0188] Example 3: Optimization of promoter screening for mbIL-15 co-expression in GC33-STAR

[0189] 3.1. Vector Construction and Virus Packaging

[0190] 1) Construction of mbIL-15-STAR vectors with different promoters

[0191] Targeting GPC3, a GC33 STAR was constructed using the antibody GC33 (SEQ ID NO:24). The STAR contains an OX40 co-stimulatory domain that connects to the C-terminus of the TRAC or TRBC constant region via a linker or directly. The constant regions of the STAR molecule are TRAC (Cys-TM) and TRBC (Cys-TM). Similar to Example 2, it was co-expressed with mbIL-15, but with different promoters (HTLV promoter (SEQ ID NO:35) and MND promoter (SEQ ID NO:34)) to drive the fusion protein expression. Simultaneously, mbIL-15 was replaced with CXCR2 (WO2022 / 179520A). The three constructed vector structures are shown in Figure 7: MND-GC33-STAR-mbIL-15, MND-GC33-STAR-CXCR2, and HTLV-GC33-STAR-CXCR2.

[0192] 2) Packaging viruses

[0193] Lentix-293T cells were divided into groups of 5 × 10 5 Inoculate cells / mL into 10cm culture dishes and transfect when the cell density reaches about 80%. The ratio of the four plasmids is PMD2.G:PRSV-Rev:PMDlg:transfer plamid = 1:1:2:4. The volume-to-mass ratio of PEI-Max to plasmid is 3:1. Change the medium after 12-16 hours and collect the virus solution after 48 hours and 72 hours.

[0194] 3) Virus titer determination

[0195] The virus was serially diluted 10-fold in 96-well plates, and then Jurkat-C5 cells with TCR knockout were incubated at 1.5 × 10⁻⁶ wells. 5 Virus cells / mL were added to the wells, centrifuged at 32°C and 1500 rpm for 90 min, and incubated for 72 h. Infection efficiency was measured by flow cytometry. Wells with infection rates between 2-30% were selected for titer calculation. Titrate (TU / mL) = 1.5 × 10⁻⁶. 4 × Positive rate ÷ Viral volume (μL) × 1000.

[0196] 3.2. Membrane expression and detection of different GPC3-STARs driven by different promoters

[0197] 1) Isolation, activation, and infection of human primary T cells

[0198] After obtaining PBMCs using the Ficoll method for cell separation and counting, they were incubated with 1.5 times the amount of CD3 / CD28 Dynabeads for 45 min, followed by incubation at a concentration of 1.2 × 10⁻⁶ cells / mL. 6 The cells were cultured at a density of 1 / mL, and after 24 hours, they were infected with the virus at an MOI of 2. After 24 hours, the medium was changed, and the cells were passaged every other day.

[0199] 2) Detection of STAR infection efficiency and co-expression efficiency under different construction strategies

[0200] The GPC3-STRAR vector was packaged into lentivirus and used to infect T cells. During a two-week continuous culture period, samples were taken at time points day 5, day 7, day 10, day 12, and day 14. Flow cytometry was used to detect the co-expression levels of STAR and different proteins on the cells. mTCRβ antibody staining detected the STAR receptor on the cell membrane, and APC antibody detection was used to detect receptor expression. As shown in Figure 8, both the HTLV and MND promoters achieved good co-expression levels of GPC3 with mbIL-15 or CXCR2 proteins.

[0201] 3.3. Comparison of in vitro functional validation of T cells expressing GPC3-STAR driven by different promoters

[0202] Three types of T cells—MND-GC33-STAR-mbIL-15, MND-GC33-STAR-CXCR2, and HTLV-GC33-STAR-CXCR2—were co-cultured with target cells Huh7-LUC-GFP at a 1:1 effector-to-target ratio. After co-culture, the killing level of the three T cells against the target cells was assessed every 24 hours. Once the target cells in the wells were completely killed, the co-cultured cells were transferred to new target cell wells for further co-culture. The killing level of the target cells in the wells was then assessed every 24 hours until no further cell killing occurred, at which point the experiment was terminated.

[0203] As shown in Figure 9, MND-GC33-STAR-mbIL-15 exhibits the strongest continuous killing capability among the three structures. For the same fusion protein, the continuous killing efficiency of the promoter MND (MND-GC33-STAR-CXCR2) is higher than that of the promoter HTLV (HTLV-GC33-STAR-CXCR2).

[0204] Comparison of cytokine secretion:

[0205] After co-culturing T cells with target cells in the killing experiment, the supernatant was collected. The secretion levels of IFN-γ and IL-2 were detected by ELISA.

[0206] During T cell activation, a large number of cytokines are released to help T cells kill target cells or promote T cell proliferation. Common cytokines include TNF-α, IFN-γ, and IL-2. After T cells are stimulated by target cells or antigens, they are collected, centrifuged, and the supernatant is collected. The TNF-α, IFN-γ, and IL-2 ELISA kits used are Human IL-2 Uncoated ELISA, Human TNF-α Uncoated ELISA, and Human IFN-γ Uncoated ELISA (catalog numbers 88-7025, 88-7346, and 88-7316, respectively). The specific steps are as follows: Dilute 10X Coating Buffer to 1X with ddH2O, add the coating antibody (250X), mix well, and then add 100 μL / well to a 96-well plate (ELISA specific). After sealing with plastic wrap, incubate overnight at 4°C. Wash three times with 1X PBST (also known as Wash Buffer, 1X PBS with 0.05% Tween 20), 260 μL / well each time. Dilute 5X ELISA / ELISPOT Diluent to 1X with ddH2O, add 200 μL / well to a 96-well plate, and incubate at room temperature for 1 hour. Wash once with PBST, and dilute the standard curves (ranges: 2–250, 4–500, 4–500). Dilute the samples 20–50 times with 1X Diluent. Add 100 μL of sample and standard curve to each well, in duplicate. Incubate at room temperature for 2 h, then wash three times with PBST. Add 1x Diluent diluted detection antibody and incubate for 1 h. Wash three times with PBST, then add 1x Diluent diluted HRP and incubate for 30 min. Wash six times, add TMB for color development (no more than 15 min), and stop the reaction with 2N H2SO4. Detect the light absorption at 450 nm.

[0207] As shown in Figure 10, co-culturing T cells with target cells (Huh7-LUC-GFP) significantly stimulated T cells to secrete IL-2γ and IFN-γ. Regarding IFN-γ cytokine concentration, STAR-T cells with the MND-GC33-STAR-mbIL-15 structure exhibited the highest IFN-γ cytokine secretion level. When comparing different promoters with the same co-expressed protein, STAR-T cells with the MND (MND-GC33-STAR-CXCR2) promoter structure showed a significantly higher IFN-γ cytokine secretion concentration than those with the HTLV (HTLV-GC33-STAR-CXCR2) promoter structure. Regarding IL-2γ cytokine concentration, STAR-T cells with both MND promoter protein structures showed comparable IL-2 cytokine secretion levels, while those with the HTLV (HTLV-GC33-STAR-CXCR2) promoter structure exhibited the lowest IL-2 cytokine secretion level.

[0208] 3.4. In vivo functional validation of STAR-T cells targeting GPC3

[0209] An NPG immunodeficient mouse model was established using NPG mice. These mice lack T cells, B cells, and NK cells, and also exhibit deficiencies in macrophages and dendritic cells. Female NPG mice aged 6–8 weeks were used in this experiment, with the weight difference between batches controlled to within 2g. Mice were housed in specific pathogen-free (SPF) individually ventilated cages, provided with a normal diet and slightly acidic drinking water to prevent pathogen contamination. All animal operations were performed after approval of the Animal Research and Use Protocol (ARP).

[0210] A tumor model was constructed using xenografting of the human Huh7-Luc / GFP cell line. Huh7 cells are a cell line that expresses the luciferase gene via a lentiviral vector. The development and changes of Huh7 tumors were monitored in real time in mice using luciferin chemiluminescence and in vivo imaging.

[0211] In this model, 3×10 6 Huh7-Luc / GFP cells were subcutaneously inoculated into 6-8 week old female NPG mice. Fluorescence signals of tumor cells in vivo were detected by in vivo imaging after intraperitoneal injection of fluorescein potassium solution into the mice. On day 6 post-Huh7-Luc / GFP cell inoculation, MND-GC33-STAR-mbIL-15, MND-GC33-STAR-CXCR2, and HTLV-GC33-STAR-CXCR2 T cells, as well as uninfected STAR T cells (MOCK-T), were infused via the tail vein. Two dose groups were set for each STAR-T cell type: 1×10⁻⁶ cells.6 / each, and the total cell count of the three groups was adjusted using MOCK-T. Then, on days -1, 5, 8, 14, 21, and 29, tumor growth, tumor fluorescence value, and weight changes were detected periodically using luciferin substrate catalytic luminescence assay.

[0212] The results are shown in Figures 11(A)-11(C). Under the condition of the same co-expressed protein but different promoters, T cells with the MND promoter (MND-GC33-STAR-CXCR2) showed better efficacy and faster, stronger tumor-killing effects than T cells with the HTLV promoter (HTLV-GC33-STAR-CXCR2). Furthermore, neither type of T cell resulted in a significant decrease in mouse body weight, indicating relatively good safety. Under the condition of the same promoter but different co-expressed proteins, T cells with mbIL-15 (MND-GC33-STAR-mbIL-15) showed better efficacy and faster, stronger tumor-killing effects than T cells with CXCR2 (MND-GC33-STAR-CXCR2). Furthermore, neither type of T cell resulted in a significant decrease in mouse body weight, indicating relatively good safety. Based on these efficacy experiments, MND was selected as the promoter for mbIL-15STAR.

[0213] 3.5. Functional study of GPC3-STAR-T cells co-expressing mbIL-15

[0214] 1) Preparation of GC33-mbIL-15 samples

[0215] T cells were enriched and activated using Thermo Fisher Scientific CD3 / CD28 magnetic beads. Cells were cultured in X-vivo15 complete medium and, approximately 24 hours after activation, transfected with viruses containing the GC33-CXCR2 sequence (MND promoter) and varying amounts of the GC33-mbIL-15 sequence (MND promoter). On day 3, the medium was replaced with fresh X-vivo15 complete medium, and cultured for another 7 days. After concentration and washing, samples were cryopreserved using CS10 cryopreservation solution with a gradient cooling process. A total of four samples were obtained; sample information is as follows:

[0216] Table 1. GC33-mbIL-15 Sample Information

[0217] 2) Experimental design for functional study of GC33-STAR-T cells co-expressing mbIL-15

[0218] Based on the fact that IL-15Rα and IL-15 can enhance cell viability through cis (cis) and trans (trans) pathways after binding with IL-2 / IL-15Rβ and γc, and activate the JAK3 pathway leading to STAT5 phosphorylation, functional studies primarily focused on STAT5 phosphorylation and cell viability under target-free and serum-free culture conditions. The experimental protocol and results are as follows:

[0219] Cell viability (survival study) research

[0220] Resuscitate the above four groups of cell samples separately, culture them using X-vivo15, and add 1×10⁶ cells / well to each well of a 24-well plate. 6 Cells were cultured at 5% CO2 at 37°C for 100 cells / well, and cell viability was observed at 0, 1, 3, 5, 8, 10, 13, 15, 20, and 22 days. The results are shown in Figure 12. Under these conditions, the GC33-CXCR2 sequence resulted in a faster decrease in cell viability compared to the GC33-mbIL-15 structure, and exhibited lower cell viability. There was no significant difference in GC33 / mbIL-15 positivity rates among samples. Conclusion: GC33-STAR-T cells co-expressing mbIL-15 have higher viability and longer survival time.

[0221] STAT5 phosphorylation study

[0222] The above four groups of samples were revived separately, and then incubated with huh-7 cells in X-vivo15 medium at a 1:1 ratio of START cells to target cells in 24-well plates for 1 hour, at a concentration of 1×10⁻⁶ cells / well. 6 Cells / well. Then, flow cytometry was used to stain pSTAT5-PE / mbIL-15Ra-APC to observe the percentage change of pSTAT5 in different sequence structures before and after incubation with target cells.

[0223] Figures 13(A)-13(B) show the expression levels of pSTAT5 in different samples before and after incubation with Huh-7 cells. Results: The pSTAT5 level in the GC33-mbIL-15 structure was higher than that in the GC33-CXCR2 structure before incubation; after incubation with Huh7 cells, the pSTAT5 level increased rapidly in all groups, and there was no correlation between pSTAT5 expression and mbIL-15 expression.

[0224] Example 4: Dual epitope LILRB4 STAR-T co-expressing mbIL-15

[0225] 4.1. Vector Construction and Virus Packaging

[0226] 1) Construction of the STAR-T vector co-expressing mbIL-15 dual epitope LILRB4

[0227] Previously, the inventors obtained LILRB4-specific nanobodies NBL4 (SEQ ID NO:27) and NBL14 (SEQ ID NO:28) using a nanobody screening platform. These STARs contain an OX40 co-stimulatory domain linked either via a linker or directly to the C-terminus of the TRAC or TRBC constant region. The constant regions of the STAR molecules are TRAC (Cys-TM) and TRBC (Cys-TM). Similar to Example 2, a dual-epitope NBL4-NBL14 STAR-T was constructed, exhibiting stronger in vitro and in vivo antitumor effects. mbIL-15 was linked to the LILRB4 STAR via furin-P2A (Figure 14).

[0228] 2) Packaging viruses

[0229] Lentix-293T cells were divided into groups of 5 × 10 5 Inoculate cells / mL into 10cm culture dishes and transfect when the cell density reaches about 80%. The ratio of the four plasmids is PMD2.G:PRSV-Rev:PMDlg:transfer plamid = 1:1:2:4. The volume-to-mass ratio of PEI-Max to plasmid is 3:1. Change the medium after 12-16 hours and collect the virus solution after 48 hours and 72 hours.

[0230] 3) Virus titer determination

[0231] The virus was serially diluted 10-fold in 96-well plates, and then Jurkat-C5 cells with TCR knockout were incubated at 1.5 × 10⁻⁶ wells. 5 Virus cells / mL were added to the wells, centrifuged at 32°C and 1500 rpm for 90 min, and incubated in an incubator. After 72 h, the infection efficiency was measured by flow cytometry. Wells with an infection rate of 2-30% were selected for titer calculation. Titrate (TU / mL) = 1.5 × 10⁻⁶. 4 × Positive rate ÷ Viral volume (μL) × 1000.

[0232] 4.2. Expression and Detection of LILRB4-STAR on Membrane

[0233] 1) Isolation, activation, and infection of human primary T cells

[0234] After obtaining PBMCs using the Ficoll method for cell separation and counting, they were incubated with 1.5 times the amount of CD3 / CD28 Dynabeads for 45 min, followed by incubation at a concentration of 1.2 × 10⁻⁶ cells / mL. 6 The cells were cultured at a density of 1 / mL, and after 24 hours, they were infected with the virus at an MOI of 2. After 24 hours, the medium was changed, and the cells were passaged every other day.

[0235] 2) STAR infection efficiency and co-expression efficiency detection

[0236] Seventy-two hours after infection, STAR infection efficiency was analyzed by detecting the mTCRβ ratio using flow cytometry after staining with BV421-anti mTCRβ antibody and APC anti-IL15Ra antibody.

[0237] 3) Protein co-expression verification

[0238] Cells, culture supernatants, and proteins of LILRB4 STAR-T cells with different structural modifications were collected, and IL-15Rα on the cell membrane surface was detected by flow cytometry, ELISA, and Western blotting, respectively.

[0239] 4.3. Killing efficiency of LILRB4 STAR-T cells co-expressing mbIL-15 against target cells

[0240] MV4-11-LUC-GFP target cells expressing the LILRB4 target were seeded in 24-well plates at a density of 4E5 / well. STAR-T cells (NLB4-NLB14 STAR-mbIL-15) were added to the target cells at STAR-positive T cell to target cell ratios of 0.2:1 and 0.6:1, with a co-culture volume of 1 mL. After 24 hours of co-culture, the co-cultured cell suspension was collected, and the LUC luminescence value was detected using a luciferase reporter gene assay kit to calculate the killing efficiency of STAR-T cells against target cells. The results (Figure 15) showed no significant difference in the 24-hour short-term killing effect between NLB4-NLB14 STAR-mbIL-15 and NLB4-NLB14 STAR under the two effector-target ratio conditions.

[0241] 4.4. In vivo efficacy evaluation of LILRB4 STAR-T co-expressing mbIL-15

[0242] This experiment will use 6-8 week old female NPG mice, with the weight difference between mice in each batch controlled within 2g. Mice will be housed in individual, ventilated cages within a specific pathogen-free (SPF) clean-barrier environment, provided with a normal diet and slightly acidic drinking water to prevent pathogen contamination. All animal operations will be performed only after approval of the Animal Protocol for Research and Use.

[0243] The effects of mbIL-15 co-expressing NLB4-NLB14 STAR cells on in vivo drug efficacy and proliferation were investigated using the MV4-11 tumor model. Fluorescently labeled MV4-11 cells (acute myeloid leukemia cells) were inoculated into NPG female mice via tail vein infusion to construct an in vivo tumor imaging model of MV4-11 cells in NPG mice. At 6-8 weeks of age, NPG female mice were intravenously infused with 2 × 10⁻⁶ cells. 6 / mouse of MV4-11-LUC-GFP target cells were infused with NLB4-NLB14 STAR-T cells co-expressing mbIL-15 on day 8 after inoculation, with NLB4-NLB14 STAR-T cells serving as the control group. The infusion dose of each STAR-T cell was 1E6 / mouse. Tumor fluorescence values ​​and body weight changes were observed after infusion (Figure 16(A)-(C)). Simultaneously, the in vivo expansion of CD3+ T and STAR-T cells and the CD4+ / CD8+ T cell ratio in peripheral blood of mice were measured (Figure 17(A)-(C)). In the MV4-11 target cell tumor model, NLB4-NLB14 STAR-mbIL-15 (i.e., LILRB4 Dual STAR-mbIL-15 in the attached figure) T cells showed better efficacy than NLB4-NLB14-STAR (i.e., LILRB4 Dual STAR in the attached figure), exhibiting faster and stronger tumor-killing effects. Furthermore, neither type of T cell resulted in a significant decrease in mouse body weight, indicating relatively good safety.

[0244] 4.5. Functional study of LILRB4-STAR-T cells co-expressing mbIL-15

[0245] 1) Preparation of LILRB4 dual-mbIL-15 samples

[0246] T cells were enriched and activated using magnetic beads from Miltenyi and Thermo Fisher Scientific, respectively. Cells were cultured in X-vivo15 complete medium. After approximately 24 hours of activation, cells were transfected with viruses containing or without the mbIL-15 sequence structure. On day 3, the medium was replaced with fresh X-vivo15 complete medium, and cultured for another 9 days. After concentration and washing, the samples were cryopreserved in CS10 cryopreservation solution using a gradient cooling method. A total of three samples were obtained. Sample information is as follows:

[0247] Table 2. Information on LILRB4 dual-mbIL-15 samples

[0248] 2) Experimental design for functional study of LILRB4 dual-STAR-T cells co-expressing mbIL-15

[0249] Since IL-15Rα and IL-15 can enhance cell viability through cis and trans pathways after binding with IL-2 / IL-15Rβ and γc, and activate the JAK3 pathway to phosphorylate STAT5, functional studies mainly focus on STAT5 phosphorylation and cell viability under target-free and serum-free culture conditions. The experimental protocol and results are as follows.

[0250] Cell viability (survival study) research

[0251] Resuscitate the above three groups of cell samples separately, culture them using X-vivo15, and add 1×10⁶ cells / well to each well of a 24-well plate. 6 Cells per well were cultured at 37°C with 5% CO2. Viability changes were observed by counting cells at days 0, 1, 3, 5, and 8. Results are shown in Figure 18. Under these culture conditions, LILRB4 cells without the mbIL-15 sequence showed a faster decline in viability and lower viability compared to those with the mbIL-15 sequence. There was no significant difference in LILRB4 / mbIL-15 positivity rates among samples. Conclusion: LILRB4 dual-STAR-T cells co-expressing mbIL-15 exhibit higher viability and longer survival time.

[0252] STAT5 phosphorylation study

[0253] The three groups of samples were revived separately and then incubated with MV-4-11 cells in X-vivo15 medium at a STAR-T cell to target cell ratio of 1:1 in 24-well plates for 1 hour, at a concentration of 1×10⁻⁶ cells / well. 6 Cells / well. Flow cytometry was then used to stain (pSTAT5-PE / mbIL-15Ra-APC) to observe the percentage change in pSTAT5 before and after incubation of STAR-T cells containing different sequence structures with target cells.

[0254] Figures 19(A)-19(B) show the expression levels of pSTAT5 in different samples before and after incubation with MV-4-11 cells. Results: The LILRB4-STAR-mbIL-15 structure showed higher pSTAT5 levels after incubation than the structure without mbIL-15.

[0255] Example 5: BCMA / LILRB4 STAR-T co-expressing mbIL-15

[0256] 5.1. Nanobodies targeting BCMA and STAR

[0257] The inventors obtained BCMA-specific nanobodies, including NBC11 (SEQ ID NO:29), NBC15 (SEQ ID NO:30), NBC16 (SEQ ID NO:31), NBC21 (SEQ ID NO:32), and NBC23 (SEQ ID NO:33), using a nanobody screening platform. These antibodies were also verified to be suitable for constructing STAR-T cells targeting BCMA, effectively killing BCMA+ target cells. Among STAR-T cells, antibody NBC11 showed the best performance.

[0258] 5.2. Construction of STAR vectors co-expressing mbIL-15 and NBC11-NLB14 and viral packaging

[0259] Similar to Example 2, an NBC11 / NBL14 STAR targeting both BCMA and LILRB4 was constructed, and mbIL-15 was linked to this STAR via furin-P2A (vector structure shown in Figure 20). The aforementioned STAR contains an OX40 co-stimulatory domain connected either via a linker or directly to the C-terminus of the TRAC or TRBC constant region. The constant regions of the STAR molecule are TRAC (Cys-TM) and TRBC (Cys-TM). The corresponding nucleic acid sequences were synthesized and assembled, and then inserted into a lentiviral vector using homologous recombination to construct the STAR-T plasmid.

[0260] Lentix-293T cells were divided into groups of 5 × 10 5 Inoculate cells / mL into 10cm culture dishes and transfect when the cell density reaches about 80%. The ratio of the four plasmids is PMD2.G:PRSV-Rev:PMDlg:transfer plamid = 1:1:2:4. The volume-to-mass ratio of PEI-Max to plasmid is 3:1. Change the medium after 12-16 hours and collect the virus solution after 48 hours and 72 hours.

[0261] After obtaining PBMCs using the Ficoll method for cell separation and counting, they were incubated with 1.5 times the amount of CD3 / CD28 Dynabeads for 45 min, followed by incubation at a concentration of 1.2 × 10⁻⁶ cells / mL. 6 Cells were cultured at a density of [insert cell density here] mL, and infected with the virus at an MOI of 4 after 24 hours. The medium was changed after 24 hours, and subsequent passages were performed every other day. 72 hours post-infection, STAR infection efficiency was analyzed by flow cytometry to detect the mTCRβ ratio using BV421-anti mTCRβ antibody, APC-antiIL-15Ra antibody, PE-LILRB4, and FITC-BCMA staining.

[0262] The results are shown in Figure 21. The co-expression efficiency of mbIL-15 in the NBC11-NLB14 STAR membrane was greater than 55%, and the co-expression efficiency of mbIL-15 protein in cells with the STAR structure membrane was greater than 96%. Meanwhile, the expression efficiencies of LILRB4 and BCMA antibodies in cells with the STAR structure membrane were measured at 53.3% and 50.7%, respectively. These results indicate that LILRB4, BCMA antibody, and mbIL-15 protein in the NBC11-NLB14 STAR-mbIL-15 structure can achieve relatively uniform and efficient membrane expression.

[0263] 5.3. Effect of co-expression of mbIL-15 on in vitro expansion of BCMA / LILRB4 STAR-T

[0264] During T cell activation, a large number of cytokines are released to help T cells kill target cells or promote T cell proliferation. The most obvious manifestation of T cell proliferation is a significant change in the number of T cells. After co-incubating T cells with target cells for 4, 8, 13, and 17 days, the cells were centrifuged, resuspended in PBS to 200 μL, and the changes in the number of positive T cells were counted by flow cytometry. Changes in T cell proliferation: Fold rate = Number of positive T cells / Initial amount of positive T cells added.

[0265] NBC11-NLB14-STAR-mbIL-15T cells with infection efficiencies M1 (MOI = 1.5) and M2 (MOI = 4) and NCI-H929 target cells were initially co-cultured with the target cells at an effector-to-target ratio of 0.8:1. The corresponding NBC11-NLB14-STAR T cells (STAR ​​vector structure shown in Figure 21) served as a control. Cells were collected at days 0, 4, 8, 13, and 17 for flow cytometry analysis. The culture medium used was cytokine-free 1640 complete medium, and the initial STAR-T cell number was 3.2 × 10⁻⁶. 5 Cells and samples at each time point were incubated independently, and the remaining co-incubated samples were partially replaced with target cells every other day. Cells used for flow cytometry analysis were first stained with anti-human CD3 antibody, and a specified volume of cells was collected and recorded before flow cytometry analysis. The number and proportion of T cells in the system were then calculated.

[0266] As shown in Figures 22(A)-(C), the proliferation curves of absolute T cells and STAR cells show that STAR-T cells co-expressing mbIL-15 expanded significantly more in vitro than STAR-T structures without mbIL-15 under the same target cell stimulation conditions. The STAR-mbIL-15 infection efficiency of NBC11-NLB14 was between 17% and 30%, and there was no significant difference in the expansion level of STAR-T cells.

[0267] 5.4. Validation of the continuous kill advantage of NBC11-NLB14 STAR co-expressing mbIL-15

[0268] The NBC11-NLB14-STAR and NBC11-NLB14-STAR-mbIL-15 vectors were expressed in T cells, with uninfected STAR T cells (MOCK-T) serving as a control. NCI-H929-luc target cells (myeloma cells) expressing BCMA and LILRB4 targets were used at a rate of 4 × 10⁻⁶. 5 The cells were seeded in 24-well plates. STAR-positive T cells and target cells were co-cultured at an effector-to-target ratio of 0.8:1. Target cell killing was assessed every 48 hours. When complete target cell killing was detected, 4 × 10⁶ cells were added to each well. 5 The NCI-H929-luc target cells were co-cultured, and the killing effect on the target cells was detected every 48 hours until the target cells were no longer killed, at which point the experiment ended.

[0269] As shown in Figure 23, the continuous kill efficiency of NBC11-NLB14-STAR-mbIL-15 is higher than that of NBC11-NLB14-STAR.

[0270] 5.5. In vivo efficacy of mbIL-15 co-expressing NBC11-NLB14 STAR mixed tumor model

[0271] This experiment used female NPG mice aged 6–8 weeks, with the weight difference between mice in each batch controlled within 2g. Mice were housed in individual, ventilated cages within a specific pathogen-free (SPF) clean-barrier environment, provided with a normal diet and slightly acidic drinking water to prevent pathogen contamination. All animal operations were performed only after approval of the Animal Protocol for Research and Use.

[0272] NCI-H929-LUC (BCMA / LILRB4 double-positive) cells, NCI-H929-LUC-LILRB4KO (BCMA single-positive) cells, and NCI-H929-LUC-BCMAKO (LILRB4 single-positive) cells were mixed at a ratio of 20%, 40%, and 40% respectively to prepare NCI-H929 mixed tumor cells. In an in vivo mouse xenograft model, the killing effect of NBC11-NLB14 STAR-T cells co-expressing mbIL-15 on mixed MM tumor cells with heterogeneous expression of BCMA / LILRB4 was verified.

[0273] The aforementioned mixed fluorescently labeled target cells were subcutaneously inoculated into 6-8 week old female NPG mice at a dose of 5E6 / mouse. On day 6 post-inoculation, NBC11-NLB14-START-mbIL-15 cells were infused via the tail vein. Simultaneously infused control STAR-T cells included: BCMA STAR-T cells recognizing only BCMA, LILRB4 STAR-T cells recognizing only LILRB4, and B2-NLB14 STAR (SEQ ID NO:79) T cells (a BCMA antibody from Legend Biotech's Cidargen Orenza) and NLB14 antibody, recognizing both BCMA and LILRB4, and a MOCK-T cell control group, all at a dose of 1.5E6 / mouse. Starting on day 5 post-T cell infusion, tumor growth was detected using a luciferin substrate-catalyzed chemiluminescence assay, and T cell expansion in mice was monitored by peripheral blood flow cytometry.

[0274] As shown in Figures 24(A)-(C), T cells targeting the dual-target BCMA-LILRB4 STAR-mbIL-15 exhibited better tumor growth inhibition compared to the control group of STAR-T cells recognizing only a single target (BCMA STAR-T, LILRB4 STAR-T) and the dual-target B2-NLB14 STAR-T cell control. In terms of in vivo T cell expansion levels, T cells expressing NBC11-NLB14 STAR-mbIL-15 also showed higher expansion levels and persistence in vivo.

[0275] This application involves the following sequences and descriptions:

Claims

1. An isolated therapeutic immune cell comprising a synthetic T-cell receptor antigen receptor (STAR) and a membrane-bound IL-15 protein (mbIL-15).

2. The therapeutic immune cell according to claim 1, wherein the mbIL-15 is a fusion protein formed by linking the extracellular domains of IL-15 and IL-15Ra (e.g., via a linker).

3. The therapeutic immune cells according to claim 2, wherein... i) IL-15 contains the amino acid sequence shown in SEQ ID NO:18; ii) The extracellular domain of IL-15Ra contains the amino acid sequence shown in SEQ ID NO:19; iii) The linker connecting the extracellular domain of IL-15Ra to IL-15 contains the amino acid sequence shown in SEQ ID NO:20; and / or iv)mbIL-15 contains the amino acid sequence shown in SEQ ID NO:

21.

4. The therapeutic immune cell according to any one of claims 1-3, wherein the STAR comprises an α chain and a β chain, the α chain comprising a first constant region, the β chain comprising a second constant region, and wherein the α chain and / or the β chain further comprises an antigen-binding region that specifically binds to a target antigen.

5. The therapeutic immune cell according to claim 4, wherein the first constant region is a natural TCRα chain constant region, for example, a natural human TCRα chain constant region or a natural mouse TCRα chain constant region; or, the first constant region is a modified TCRα chain constant region.

6. The therapeutic immune cell of claim 5, wherein the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, wherein, relative to the wild-type mouse TCRα chain constant region, the amino acid at position 48, for example threonine T, is mutated to cysteine ​​C.

7. The therapeutic immune cell according to claim 5 or 6, wherein the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, wherein, relative to the wild-type mouse TCRα chain constant region, the amino acid at position 112, such as serine S, is replaced with leucine L, the amino acid at position 114, such as methionine M, is replaced with isoleucine I, and the amino acid at position 115, such as glycine G, is replaced with valine V.

8. The therapeutic immune cell according to any one of claims 5-7, wherein the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, wherein, relative to the wild-type mouse TCRα chain constant region, the amino acid at position 6, such as E, is replaced by D, the amino acid at position 13, K, is replaced by R, and the amino acids at positions 15-18 are deleted.

9. The therapeutic immune cell according to any one of claims 5-8, wherein the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, wherein, relative to the wild-type mouse TCRα chain constant region, the amino acid at position 48, such as threonine (T), is mutated to cysteine ​​(C), the amino acid at position 112, such as serine (S), is mutated to leucine (L), the amino acid at position 114, such as methionine (M), is mutated to isoleucine (I), and the amino acid at position 115, such as glycine (G), is mutated to valine (V).

10. The therapeutic immune cell according to any one of claims 5-9, wherein the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, wherein, relative to the wild-type mouse TCRα chain constant region, the amino acid at position 6, such as E, is replaced by D; the amino acid at position 13, K, is replaced by R; the amino acids at positions 15-18 are deleted; the amino acid at position 48, such as threonine T, is mutated to cysteine ​​C; the amino acid at position 112, such as serine S, is replaced by leucine L; the amino acid at position 114, such as methionine M, is replaced by isoleucine I; and the amino acid at position 115, such as glycine G, is replaced by valine V.

11. The therapeutic immune cell according to any one of claims 5-10, wherein the TCRα chain constant region is a non-intracellular region of the constant region relative to the wild-type TCRα chain constant region, for example, lacking amino acids 136-137.

12. The therapeutic immune cell according to any one of claims 5-11, wherein the modified TCRα chain constant region is derived from the mouse TCRα chain constant region, and which, relative to the wild-type mouse TCRα chain constant region, lacks the intracellular region of the constant region, for example, lacking amino acids 136-137.

13. The therapeutic immune cell according to any one of claims 5-12, wherein the modified TCRα chain constant region comprises an amino acid sequence shown in one of SEQ ID NO: 3-7.

14. The therapeutic immune cell according to any one of claims 4-13, wherein the second constant region is a natural TCRβ chain constant region, for example, a natural human TCRβ chain constant region or a natural mouse TCRβ chain constant region; or, the second constant region is a modified TCRβ chain constant region.

15. The therapeutic immune cell of claim 14, wherein the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, wherein, relative to the wild-type mouse TCRβ chain constant region, the amino acid at position 56, for example serine S, is mutated to cysteine ​​C.

16. The therapeutic immune cell according to claim 14 or 15, wherein the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, wherein, relative to the wild-type mouse TCRβ chain constant region, the amino acid at position 3, such as R, is replaced by K; the amino acid at position 6, such as T, is replaced by F; the amino acid at position 9, such as K, is replaced by E; the amino acid at position 11, such as S, is replaced by A; the amino acid at position 12, such as L, is replaced by V; and the amino acids at positions 17 and 21-25 are deleted.

17. The therapeutic immune cell according to any one of claims 14-16, wherein the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, wherein, relative to the wild-type mouse TCRβ chain constant region, the amino acid at position 56, such as serine S, is mutated to cysteine ​​C; the amino acid at position 3, such as R, is substituted with K; the amino acid at position 6, such as T, is substituted with F; the amino acid at position 9, K, is substituted with E; the amino acid at position 11, S, is substituted with A; the amino acid at position 12, L, is substituted with V; and the amino acids at positions 17 and 21-25 are deleted.

18. The therapeutic immune cell according to any one of claims 14-17, wherein the intracellular region of the TCRβ chain constant region is missing relative to the wild-type TCRβ chain constant region, for example, the intracellular region is missing amino acids 167-172.

19. The therapeutic immune cell according to any one of claims 14-18, wherein the modified TCRβ chain constant region is derived from the mouse TCRβ chain constant region, and which, relative to the wild-type mouse TCRβ chain constant region, lacks the intracellular region of the constant region, for example, lacking amino acids 167-172.

20. The therapeutic immune cell according to any one of claims 4-19, wherein the modified TCRβ chain constant region comprises the amino acid sequence shown in one of SEQ ID NO:10-14.

21. The therapeutic immune cells according to any one of claims 4-20, wherein i) The first constant region contains the amino acid sequence shown in SEQ ID NO:3, and the second constant region contains the amino acid sequence shown in SEQ ID NO:10; ii) The first constant region contains the amino acid sequence shown in SEQ ID NO:6, and the second constant region contains the amino acid sequence shown in SEQ ID NO:10; iii) The first constant region contains the amino acid sequence shown in SEQ ID NO:3, and the second constant region contains the amino acid sequence shown in SEQ ID NO:13; or iv) The first constant region contains the amino acid sequence shown in SEQ ID NO:6, and the second constant region contains the amino acid sequence shown in SEQ ID NO:

13.

22. The therapeutic immune cell according to any one of claims 4-21, wherein the α chain and / or β chain, preferably the α chain and β chain, are connected to at least one exogenous intracellular functional domain at their C-terminus, such as the intracellular domain of a co-stimulatory molecule, preferably the intracellular domain of OX40, and more preferably, the intracellular domain of OX40 comprises the amino acid sequence of SEQ ID NO:

15.

23. The therapeutic immune cell of claim 22, wherein the exogenous intracellular functional domain is directly or via a adapter connected to the C-terminus of the constant region of the α chain and / or β chain (preferably α chain and β chain); preferably, the exogenous intracellular functional domain is connected via a adapter to the C-terminus of the constant region of the α chain and / or β chain (preferably α chain and β chain) missing in the intracellular region; preferably, the adapter is a (G4S)n adapter, where n represents an integer from 1 to 10, preferably, n is 3.

24. The therapeutic immune cell of claim 23, wherein the first constant region is a modified TCRα chain constant region derived from the mouse TCRα chain constant region, and relative to the wild-type mouse TCRα chain constant region, the amino acid at position 48, for example threonine T, is mutated to cysteine ​​C; the amino acid at position 112, for example serine S, is mutated to leucine L; the amino acid at position 114, for example methionine M, is mutated to isoleucine I; the amino acid at position 115, for example glycine G, is mutated to valine V; and the α chain comprises an intracellular domain of OX40 connected to the C-terminus of the constant region (e.g., directly or via a linker, for example (G4S)n linker, where n represents an integer from 1 to 10, preferably n is 3); and The second constant region is a modified TCRβ chain constant region derived from the mouse TCRβ chain constant region, wherein the amino acid at position 56, for example serine S, is mutated to cysteine ​​C relative to the wild-type mouse TCRβ chain constant region, and the β chain contains an intracellular domain of OX40 connected to the C-terminus of the constant region (e.g., directly or via a linker, such as (G4S)n linker, where n represents an integer from 1 to 10, preferably n is 3).

25. The therapeutic immune cell according to any one of claims 4-24, wherein the target antigen is a disease-associated antigen, for example... The target antigen is a cancer-associated antigen, such as selected from the following cancer-associated antigens: GPC3 (phosphatidylinositol proteoglycan 3), BCMA, mesothelin, LILRB4, CD16, CD64, CD78, CD96, CLL1, CD7, CD70, CD38, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2 / neu), Claudin18.2, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), and epidermal growth factor receptor. (EGFR), EGFR variant III (EGFRvIII), CD19, CD276, CD19, CD22, CD20, CD30, CD40, disialiacoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alpha fetal globulin (AFP), exogenous lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate enzyme-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, Prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, liver glycoside B2, CD22, insulin-like growth factor (IGF1)-I, IGF-II, IGFI receptor, major histocompatibility complex (MHC) molecule presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor matrix antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, A1 domain (TnC) of tendinin-C A1), fibroblast-associated protein (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, Foxp3, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endothelial factors, major histocompatibility complex (MHC) molecules, TNFRSF17, SLAMF7, GPRC5D, FKBP11, KAMP3, ITGA8, and FCRL5; or The target antigen is an antigen derived from a pathogen or a surface antigen of cells infected by the pathogen, such as RSVF (prevention of respiratory syncytial virus), PA (inhalation anthrax), CD4 (HIV infection); or The target antigens are antigens associated with other diseases, such as CD3 (involved in transplant rejection), CD25 (involved in acute kidney transplant rejection), C5 (involved in paroxysmal nocturnal hemoglobinuria), IL-1β (involved in cold pyridine-associated periodic syndrome), RANKL (involved in cancer-related bone injury), von Willebrand factor (involved in adult acquired thrombotic thrombocytopenic purpura), plasma kallikrein (involved in angioedema), calcitonin gene-related peptide receptor (involved in adult migraine), and FGF23 (involved in X-linked hypophosphatemia).

26. The therapeutic immune cells according to any one of claims 4-25, wherein i) The α chain comprises a first antigen-binding region and a first constant region, and the β chain comprises a second constant region; ii) The α chain contains a first constant region, and the β chain contains a second antigen-binding region and a second constant region; or iii) The α chain comprises a first antigen-binding region and a first constant region, and the β chain comprises a second antigen-binding region and a second constant region.

27. The therapeutic immune cell of claim 26, wherein the first antigen-binding region and the second antigen-binding region each independently or in combination specifically bind to the target antigen.

28. The therapeutic immune cell of claim 26 or 27, wherein the first antigen-binding region comprises a heavy chain variable region of an antibody that specifically binds to a target antigen, and the second antigen-binding region comprises a light chain variable region of the antibody; or, the first antigen-binding region comprises a light chain variable region of an antibody that specifically binds to a target antigen, and the second antigen-binding region comprises a heavy chain variable region of the antibody.

29. The therapeutic immune cell of claim 26 or 27, wherein the first antigen-binding region comprises a single-chain antibody (e.g., scFv) or a single-domain antibody that specifically binds to the target antigen; and / or the second antigen-binding region comprises a single-chain antibody or a single-domain antibody that specifically binds to the target antigen.

30. The therapeutic immune cell of claim 29, wherein the first antigen-binding region and the second antigen-binding region bind the same target antigen.

31. The therapeutic immune cell of claim 30, wherein the first antigen-binding region and the second antigen-binding region bind different regions (e.g., different epitopes) of the same target antigen; or the first antigen-binding region and the second antigen-binding region bind different target antigens.

32. The therapeutic immune cell according to any one of claims 4-31, wherein the antigen-binding region comprises the heavy chain variable region shown in SEQ ID NO:22 and / or the light chain variable region shown in SEQ ID NO:23, thereby the STAR targets GPC3.

33. The therapeutic immune cells according to claim 32, wherein in i) The first antigen-binding region includes the heavy chain variable region shown in SEQ ID NO:22, and the second antigen-binding region includes the light chain variable region shown in SEQ ID NO:23; ii) The first antigen-binding region includes the light chain variable region shown in SEQ ID NO:23, and the second antigen-binding region includes the heavy chain variable region shown in SEQ ID NO:22; iii) The first antigen-binding region comprises the heavy chain variable region shown in SEQ ID NO:22 and the light chain variable region shown in SEQ ID NO:23, for example, the first antigen-binding region comprises the single-chain antibody sequence shown in SEQ ID NO:24; or iv) The second antigen-binding region includes the heavy chain variable region shown in SEQ ID NO:22 and the light chain variable region shown in SEQ ID NO:

23. For example, the second antigen-binding region includes the single-chain antibody sequence shown in SEQ ID NO:

24.

34. The therapeutic immune cell according to any one of claims 4-31, wherein the antigen-binding region comprises a single-domain antibody sequence selected from SEQ ID NO:25 or 26, thereby wherein the STAR targets mesothelin (MSLN).

35. The therapeutic immune cell of claim 34, wherein the first antigen-binding region or the second antigen-binding region, preferably the second antigen-binding region comprises a single-domain antibody sequence selected from SEQ ID NO: 25 or 26.

36. The therapeutic immune cells according to claim 34, wherein... i) The first antigen-binding region contains the single-domain antibody sequence of SEQ ID NO:25, and the second antigen-binding region contains the single-domain antibody sequence of SEQ ID NO:26; or ii) The first antigen-binding region contains the single-domain antibody sequence of SEQ ID NO:26, and the second antigen-binding region contains the single-domain antibody sequence of SEQ ID NO:

25.

37. The therapeutic immune cell according to any one of claims 4-31, wherein the antigen-binding region comprises a single-domain antibody sequence selected from SEQ ID NO:27 or 28, thereby the STAR targets LILRB4.

38. The therapeutic immune cell of claim 37, wherein the first antigen-binding region or the second antigen-binding region, preferably the second antigen-binding region, comprises a single-domain antibody sequence selected from SEQ ID NO: 27 or 28.

39. The therapeutic immune cells according to claim 37, wherein... i) The first antigen-binding region contains the single-domain antibody sequence of SEQ ID NO:27, and the second antigen-binding region contains the single-domain antibody sequence of SEQ ID NO:28; or ii) The first antigen-binding region contains the single-domain antibody sequence of SEQ ID NO:28, and the second antigen-binding region contains the single-domain antibody sequence of SEQ ID NO:

27.

40. The therapeutic immune cell according to any one of claims 4-31, wherein the antigen-binding region comprises a single-domain antibody sequence selected from SEQ ID NO:29-33, thereby the STAR targets BCMA.

41. The therapeutic immune cells according to claim 40, wherein... i) The first antigen-binding region or the second antigen-binding region, preferably the second antigen-binding region comprising a single-domain antibody sequence selected from SEQ ID NO:29-33; or ii) The first antigen-binding region contains a single-domain antibody sequence selected from SEQ ID NO:29-33, and the second antigen-binding region contains another single-domain antibody sequence selected from SEQ ID NO:29-33.

42. The therapeutic immune cell according to any one of claims 4-31, wherein the first antigen-binding region comprises a single-domain antibody sequence selected from SEQ ID NO:27 or 28, and the second antigen-binding region comprises a single-domain antibody sequence selected from SEQ ID NO:29-33, thereby the STAR targets LILRB4 and BCMA; or The first antigen-binding region contains a single-domain antibody sequence selected from SEQ ID NO:29-33, and the second antigen-binding region contains a single-domain antibody sequence selected from SEQ ID NO:27 or 28, thereby the STAR targets LILRB4 and BCMA.

43. The therapeutic immune cell of claim 42, wherein the first antigen-binding region comprises a single-domain antibody sequence of SEQ ID NO:28, and the second antigen-binding region comprises a single-domain antibody sequence of SEQ ID NO:29, wherein the STAR targets LILRB4 and BCMA.

44. The therapeutic immune cell of claim 1, wherein the STAR comprises an α chain and a β chain, and wherein... i) The α chain contains the amino acid sequence shown in SEQ ID NO:39, and the β chain contains the amino acid sequence shown in SEQ ID NO:38; ii) The α chain contains the amino acid sequence shown in SEQ ID NO:41, and the β chain contains the amino acid sequence shown in SEQ ID NO:40; iii) The α chain contains the amino acid sequence shown in SEQ ID NO:43, and the β chain contains the amino acid sequence shown in SEQ ID NO:42; or iv) The α chain contains the amino acid sequence shown in SEQ ID NO:45, and the β chain contains the amino acid sequence shown in SEQ ID NO:44; or v) The α chain contains the amino acid sequence shown in SEQ ID NO:83, and the β chain contains the amino acid sequence shown in SEQ ID NO:

84.

45. The therapeutic immune cell according to any one of claims 1-44, wherein the immune cell is a T cell or an NK cell, preferably a T cell.

46. ​​An expression vector comprising the coding sequence of a synthetic T-cell receptor antigen receptor (STAR) as defined in any one of claims 1-45 and the coding sequence of a membrane-bound IL-15 protein (mbIL-15) as defined in any one of claims 1-45.

47. The expression vector of claim 46, wherein the expression vector comprises a) A coding nucleotide sequence comprising a fusion polypeptide of the α chain of the STAR, the β chain of the STAR, and the mbIL-15 linked by a self-cleaving peptide, preferably, the coding nucleotide sequence being operatively linked to an MND promoter; or b) comprising the encoding nucleotide sequence of a fusion polypeptide of the α and β chains of the STAR linked by a self-cleaving peptide, and the encoding nucleotide sequence of mbIL-15, wherein the encoding nucleotide sequence of the α and β chain fusion polypeptide and the encoding nucleotide sequence of mbIL-15 are each independently operatively linked to a promoter, or the encoding nucleotide sequence of the α and β chain fusion polypeptide and the encoding nucleotide sequence of mbIL-15 are linked via an internal ribosome entry site (IRES), thereby achieving co-expression of the STAR and the mbIL-15.

48. The expression vector according to claim 47, wherein the self-cleaving peptide is a 2A peptide, preferably, the self-cleaving peptide is a Furin-2A peptide, such as the Furin-P2A peptide shown in SEQ ID NO:

17.

49. The expression vector according to claim 47 or 48, wherein i) The fusion polypeptide comprises, from N-terminus to C-terminus, the β-chain, a self-cleaving peptide such as Furin-P2A, and the α-chain; or ii) The fusion polypeptide comprises, from the N-terminus to the C-terminus, the β-chain, the self-cleaving peptide such as Furin-P2A, the α-chain, the self-cleaving peptide such as Furin-P2A, and the mbIL-15.

50. The expression vector according to any one of claims 47-49, wherein the α chain, the β chain, and / or the mbIL-15 encoded by the expression vector further each comprises an N-terminal signal peptide, for example, i) The α chain and / or the β chain contains a GM-CSF signal peptide at its N-terminus, and / or, ii) The mbIL-15 contains an IgE signal peptide at its N-terminus, such as the IgE signal peptide shown in SEQ ID NO:

36.

51. A method for preparing therapeutic immune cells, comprising: Step 1) Provide initial immune cells; Step 2) Introduce the expression vector according to any one of claims 46-50 into the initiating immune cells; and Step 3) Harvest the immune cells obtained in Step 2).

52. The method according to claim 51, wherein the initiating immune cell is a T cell or an NK cell, preferably a T cell.

53. A therapeutic immune cell, such as a T cell, comprising an expression vector according to any one of claims 46-50, or an expression vector according to any one of claims 46-50 or a method according to any one of claims 51-52.

54. A pharmaceutical composition comprising a therapeutic immune cell according to any one of claims 1-46 and 53 and / or an expression vector according to any one of claims 46-50, and a pharmaceutically acceptable carrier, preferably for treating a disease in a subject.

55. Use of the therapeutic immune cells according to any one of claims 1-46 and 53, the expression vector according to any one of claims 46-50, and / or the pharmaceutical composition according to claim 54 in the preparation of a medicament for treating a disease in a subject.

56. A method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of a therapeutic immune cell according to any one of claims 1-46 and 53, an expression vector according to any one of claims 46-50, and / or a pharmaceutical composition according to claim 54.

57. The pharmaceutical composition of claim 54, the use of claim 55, or the method of claim 56, wherein the disease is selected from cancer, pathogen infection, cardiovascular disease, diabetes, neurological disease, post-transplant rejection, and autoimmune disease.

58. The pharmaceutical composition, use, or method of claim 57, wherein the cancer is selected from myeloma (e.g., multiple myeloma (MM), particularly relapsed or refractory multiple myeloma (RRMM)), lung cancer such as squamous cell carcinoma of the lung (SqCC), ovarian cancer, colon cancer, rectal cancer, colorectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer such as hepatocellular carcinoma, lymphoma, hematologic malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, laryngeal cancer, cervical cancer, endometrial tumor, osteosarcoma, bone cancer, pancreatic cancer, skin cancer, prostate cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, etc. Small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), pediatric solid tumors such as embryonal tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and combinations of the aforementioned cancers.

59. The pharmaceutical composition, use, or method of claim 57, wherein the autoimmune disease is selected from systemic lupus erythematosus (SLE), myositis, scleroderma, Sjögren's syndrome, autoimmune hemolytic anemia, and rheumatoid arthritis.

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