Antibody targeting BCMA and use thereof

Abstract

The present invention belongs to the field of biomedicine. Specifically, provided are an antibody targeting BCMA and the use thereof. More specifically, provided are an antibody targeting BCMA, STAR and / or CAR derived from the antibody, a therapeutic immune cell comprising the STAR and / or CAR, and the use thereof in the treatment of diseases.

Description

Antibodies targeting BCMA and their applications Technical Field

[0001] This invention belongs to the field of biomedicine. Specifically, this invention provides an antibody targeting BCMA and its application. More specifically, this invention provides an antibody targeting BCMA, STAR and / or CAR derived from said antibody, therapeutic immune cells containing said STAR and / or CAR, and their application in disease treatment. Background Technology

[0002] B-cell maturation antigen (BCMA, also known as CD269 or TNFRSF17) is a member of the tumor necrosis factor receptor superfamily (TNFRSF). BCMA is a type III transmembrane protein containing a cysteine-rich domain (CRD) characteristic of TNFR family members within its extracellular domain (ECD). This domain forms a ligand-binding motif. BCMA ligands include B-cell activating factor (BAFF) and B-cell proliferation-inducing ligand (APRIL). APRIL binds to BCMA with a higher affinity, promoting tumor cell proliferation.

[0003] BCMA is primarily expressed on the surface of mature B cells, i.e., plasma cells, and is not expressed in normal hematopoietic stem cells and non-hematopoietic tissues. BCMA signaling is essential for the survival of long-acting bone marrow plasma cells, but not for overall B cell homeostasis. Surface BCMA can be cleaved by γ-secretase and detached, producing soluble BCMA (sBCMA), which may reduce surface BCMA signaling by blocking BAFF / APRIL ligand binding. Preclinical models and human tumors have shown that BCMA is overexpressed in multiple myeloma (MM) cells, upregulating both classical and non-classical NF-κB signaling, promoting MM cell growth, survival, and adhesion, and inducing osteoclast activation, angiogenesis, metastasis, and immunosuppression. BCMA expression has become an important biomarker for MM diagnosis. Furthermore, serum sBCMA levels are elevated in MM patients, positively correlated with the number of MM cells in the bone marrow, and its concentration changes are closely related to MM prognosis and treatment response.

[0004] CAR-T cells targeting BCMA, such as Abecma, have been used clinically to treat multiple myeloma. However, this therapy is prone to causing serious toxic side effects such as cytokine release syndrome (CRS) and neurotoxicity (NT). Therefore, better treatment methods for multiple myeloma are still needed.

[0005] Based on the high similarity between the extracellular region of TCR and the Fab domain of antibody, the variable region sequence of TCR is replaced with the variable region sequence of antibody (such as scFv), thereby obtaining a synthetic T cell receptor and antigen receptor (STAR). STAR combines the advantages of TCR and CAR, possessing both the specificity of antibody and the superior signal transduction function of natural TCR. It can mediate complete T cell activation, and has significant improvements in safety and efficacy, thus becoming a promising new type of cell immunotherapy.

[0006] Providing new and more effective antibodies targeting BCMA and combining them with CAR or STAR technologies could offer a more effective approach to multiple myeloma. Summary of the Invention

[0007] In one aspect, the present invention provides a single-domain antibody that specifically binds to BCMA, comprising CDR1, CDR2 and CDR3 from any one of SEQ ID NO:4, 8, 12, 16 and 20.

[0008] In another aspect, the present invention provides isolated nucleic acid molecules that encode a single-domain antibody that specifically binds to BCMA according to the present invention.

[0009] In another aspect, the present invention also provides an expression vector for expressing the single-domain antibody of the present invention, which comprises the nucleic acid molecule encoding the single-domain antibody of the present invention that specifically binds to BCMA.

[0010] In another aspect, the present invention also provides host cells for preparing the single-domain antibodies of the present invention, which are transformed by the nucleic acid molecules or expression vectors described above in the present invention.

[0011] In another aspect, the present invention provides a method for generating a single-domain antibody that specifically binds to BCMA, comprising:

[0012] (i) The host cells of the present invention are cultured under conditions suitable for expression of the nucleic acid molecules or expression vectors, and

[0013] (ii) Isolate and purify a single-domain antibody that specifically binds to BCMA expressed by the host cells.

[0014] In another aspect, the present invention provides a synthetic T-cell receptor antigen receptor (STAR) targeting BCMA, comprising an antigen-binding region that specifically binds to BCMA, preferably wherein the antigen-binding region specifically binds to BCMA comprises a single-domain antibody that specifically binds to BCMA as described in the present invention.

[0015] In another aspect, the present invention provides a chimeric antigen receptor (CAR) targeting BCMA, comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain comprises a single-domain antibody that specifically binds to BCMA as described in the present invention.

[0016] In another aspect, the present invention provides an isolated therapeutic immune cell comprising the STAR or CAR of the present invention, preferably, the therapeutic immune cell further comprising membrane-bound IL-15 protein (mbIL-15).

[0017] In another aspect, the present invention provides an expression vector containing the coding sequence of the STAR or CAR of the present invention, preferably, the expression vector further containing the coding sequence of a membrane-bound IL-15 protein (mbIL-15).

[0018] In another aspect, the present invention provides a method for preparing therapeutic immune cells, comprising:

[0019] Step 1) Provide initial immune cells;

[0020] Step 2) Introduce the expression vector of the present invention containing the coding sequence of the STAR or CAR into the initiating immune cells; and

[0021] Step 3) Harvest the immune cells obtained in Step 2).

[0022] In another aspect, the present invention provides therapeutic immune cells, such as T cells, that can be obtained or acquired through the expression vector containing the coding sequence of the STAR or CAR of the present invention or the method for preparing therapeutic immune cells of the present invention.

[0023] In another aspect, the present invention provides a pharmaceutical composition comprising the single-domain antibody of the present invention, the therapeutic immune cells of the present invention, and / or the expression vector of the present invention, and a pharmaceutically acceptable vector.

[0024] In another aspect, the present invention provides the use of the single-domain antibody of the present invention, the therapeutic immune cell of the present invention, the expression vector of the present invention, and / or the pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease in a subject.

[0025] Preferably, the disease is a BCMA-related disease, such as a BCMA-related cancer, preferably a myeloma, such as multiple myeloma (MM), especially relapsed or refractory multiple myeloma (RRMM); or an autoimmune disease, such as systemic lupus erythematosus (SLE), myositis, scleroderma, or rheumatoid arthritis.

[0026] 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 single-domain antibody of the present invention, the therapeutic immune cells of the present invention, the expression vector of the present invention, and / or the pharmaceutical composition of the present invention.

[0027] Preferably, the disease is a BCMA-related disease, such as a BCMA-related cancer. More preferably, the cancer is myeloma, such as multiple myeloma (MM), especially relapsed or refractory multiple myeloma (RRMM); or an autoimmune disease, such as systemic lupus erythematosus (SLE), myositis, scleroderma, Sjögren's syndrome, autoimmune hemolytic anemia, or rheumatoid arthritis. Attached Figure Description

[0028] Figure 1 shows the BCMAs-Fc affinity assay curves. A is the affinity assay curve and fitted curve for antibody NBC11-Fc; B is the affinity assay curve and fitted curve for antibody NBC15-Fc; C is the affinity assay curve and fitted curve for antibody NBC16-Fc; D is the affinity assay curve and fitted curve for antibody NBC21-Fc; E is the affinity assay curve and fitted curve for antibody NBC23-Fc.

[0029] Figure 2 shows the competitive binding of antibody epitopes.

[0030] Figure 3 shows the MPA specificity detection of BCMA nanobodies. A is the MPA specificity detection of NBC11-Fc antibody; B is the MPA specificity detection of NBC15-Fc antibody; C is the MPA specificity detection of NBC16-Fc antibody; D is the MPA specificity detection of NBC21-Fc antibody; E is the MPA specificity detection of NBC23-Fc antibody.

[0031] Figure 4 shows different STAR structures based on BCMA antibodies.

[0032] Figure 5. In vitro functional screening of BCMA-STAR cells for Raji-luc-BCMA (Raji-Luc cells overexpressing BCMA) targeting BCMA.

[0033] Figure 6 shows the cytokine secretion of BCMA-STAR-T cells in response to Raji-luc-BCMA (Raji-Luc cells overexpressing BCMA), which targets BCMA.

[0034] Figure 7. In vivo functional evaluation of BCMA-STAR-T cells in a Raji-luc-BCMA mouse tumor model overexpressing BCMA. A is the in vivo fluorescence imaging of the mouse; B is the tumor fluorescence value; C is the mouse body weight.

[0035] Figure 8 shows the in vitro upper membrane expression of dual-target CD19-BCMA STAR detected by flow cytometry.

[0036] Figure 9. In vitro killing effect of CD19-BCMA STAR-T cells on target cells. A shows the in vitro killing effect of CD19-BCMA STAR-T cells on Raji-luc-CD19KO-BCMA target cells expressing only BCMA at E:T = 0.6:1; B shows the in vitro killing effect of CD19-BCMA STAR-T cells on Raji-luc target cells expressing only CD19 at E:T = 0.6:1.

[0037] Figure 10 shows the in vivo functional validation of CD19-BCMA STAR-T in a mouse tumor model of Raji-luc-CD19KO-BCMA target cells expressing only BCMA. A is the in vivo fluorescence imaging of the mouse; B is the tumor fluorescence value; C is the mouse body weight.

[0038] Figure 11 shows the in vitro upper membrane expression of LILRB4-BCMA STAR with different BCMA antibodies.

[0039] Figure 12. In vitro killing effect of LILRB4-BCMA STAR-T cells on target cells. A shows the in vitro killing effect of LILRB4-BCMA STAR-T cells on NCI-H929-LILRB4KO target cells at E:T = 0.5:1; B shows the in vitro killing effect of LILRB4-BCMA STAR-T cells on OCI-AML3-luc target cells at E:T = 0.5:1.

[0040] Figure 13 shows the cytokine secretion of LILRB4-BCMA STAR-T cells in response to target cells. A shows the cytokine secretion of LILRB4-BCMA STAR-T cells in response to NCI-H929-luc-LILRB4KO target cells; B shows the cytokine secretion of LILRB4-BCMA STAR-T cells in response to OCI-AML3-Luc target cells.

[0041] Figure 14 shows the in vivo functional validation of LILRB4-BCMASTAR-T in a Raji-luc-CD19KO-BCMA target cell mouse tumor model. A is the in vivo fluorescence imaging of mice; B is the tumor fluorescence value; C is the mouse body weight.

[0042] Figure 15 shows the specific killing effect of LILRB4-BCMA STAR-T on MM lineage target cells. A shows the specific killing effect of LILRB4-BCMA STAR-T on OPM2-LUC target cells; B shows the specific killing effect of LILRB4-BCMA STAR-T on U266B1-CFSE target cells; C shows the specific killing effect of LILRB4-BCMA STAR-T on MM.1S-LUC target cells; D shows the specific killing effect of LILRB4-BCMA STAR-T on PRMI8226-LUC target cells.

[0043] Figure 16 shows the in vitro upper membrane expression of LILRB4-BCMA STAR-mbIL15 with different BCMA antibodies as detected by flow cytometry.

[0044] Figure 17. This shows the validation of T cells expressing NBC11-NLB14 STAR-mbIL-15 and T cells expressing NBC11-NLB14 STAR under NCI-H929 target cell stimulation at M1 (MOI=1.5) and M2 (MOI=4) infection efficiencies. A represents the proportion of STAR-T cells; B represents total T cell expansion; C represents STAR-T cell expansion.

[0045] Figure 18 shows the continuous killing of target cells NCI-H929-LUC by T cells expressing NBC11-NLB14 STAR-mbIL-15 and NBC11-NLB14 STAR at E:T = 0.8:1.

[0046] Figure 19. Demonstration of the function of T cells with different BCMA antibodies, LILRB4-BCMA STAR-mbIL15 and LILRB4-BCMA STAR, in the H929-luc-LILRB4KO tumor model mouse. A: In vivo fluorescence imaging of mice; B: Mouse body weight; C: Tumor fluorescence value; D: Tumor volume of mice.

[0047] Figure 20 shows the functional validation of NBC11-NLB14 STAR-mbIL15 and NBC11-NLB14 STAR T cells in mice with the NCI-H929 mixed tumor model. A is the in vivo fluorescence imaging of mice; B is the tumor fluorescence value; C is the mouse body weight.

[0048] Figure 21. Flow cytometry detection of the upper membrane expression level of BCMA-LILRB4 CAR on cells.

[0049] Figure 22. Short-term and continuous killing efficiency of dual-target BCMA-LILRB4 CAR-T cells against NCI-H929-LUC target cells. A represents short-term killing; B represents continuous killing. Detailed Implementation

[0050] definition

[0051] 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.

[0052] 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.”

[0053] 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.

[0054] 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).

[0055] 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.

[0056] 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.

[0057] 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).

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] For example, as shown in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999), the amino acid residues used for the VHH domain of camelids can be numbered according to the general numbering method for VH domains given by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). However, alternative methods for numbering the amino acid residues of VH domains are known in the art, and these alternative methods can be similarly applied to VHH domains. For example, the Chothia CDR refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). The AbM CDR represents a compromise between the Kabat hypervariable region and the Chothia structural loop, and is used in Oxford Molecular's AbM antibody modeling software. The “Contact” CDR is based on the analysis of the available crystal structure of the complex.

[0063] The VHH domain, derived from the Camelidae family, can be "humanized" (also referred to herein as "sequence optimization," which, in addition to humanization, can also encompass other modifications to the sequence by one or more mutations that provide improved VHH properties, such as removing potential post-translational modification sites) by replacing one or more amino acid residues in the original VHH sequence with one or more amino acid residues present at the corresponding positions in the VH domain of a conventional human 4-chain antibody. Humanized VHH domains may contain one or more fully human framework regions. Humanization can be accomplished using protein surface amino acid resurfacing and / or CDR grafting to a universal framework.

[0064] Generally, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein (e.g., the antibody of the present invention) can bind to. Specificity can be determined based on the affinity and / or cohesion of the antigen-binding protein. Affinity, expressed as the dissociation equilibrium constant (KD) between the antigen and the antigen-binding protein, is a measure of the strength of binding between the epitope and the antigen-binding site on the antigen-binding protein: the smaller the KD value, the stronger the binding between the epitope and the antigen-binding protein (or, affinity can also be expressed as the association constant (KA), which is 1 / KD). As those skilled in the art will understand, affinity can be determined in a known manner depending on the specific antigen of interest. Affinity is a measure of the strength of binding between an antigen-binding protein (e.g., an antibody) and the associated antigen. Affinity relates to both the affinity between the antigen and the antigen-binding site on the antigen-binding protein and the number of associated binding sites present on the antigen-binding protein.

[0065] 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".

[0066] 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. In some preferred embodiments, the polypeptide of this invention comprises the GM-CSF signal peptide shown in SEQ ID NO:82. In some preferred embodiments, the polypeptide of this invention comprises the IgE signal peptide shown in SEQ ID NO:83.

[0067] 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.

[0068] 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), midway, or downstream (3' non-coding) 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, the hEF1a promoter, and the MND promoter.

[0069] 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.

[0070] 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).

[0071] 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.

[0072] Single-domain antibody that specifically binds to BCMA

[0073] In one aspect, the present invention provides a single-domain antibody that specifically binds to BCMA, comprising CDR1, CDR2, and CDR3 from any one of SEQ ID NO:4, 8, 12, 16, and 20. The CDRs may be Kabat CDR, AbM CDR, Chothia CDR, or Contact CDR. In some embodiments, the CDR is a Kabat CDR.

[0074] In some embodiments, the single-domain antibody that specifically binds to BCMA comprises a group of CDR1, CDR2, and CDR3 selected from the following:

[0075] (1) CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3;

[0076] (2) CDR1 shown in SEQ ID NO:5, CDR2 shown in SEQ ID NO:6, and CDR3 shown in SEQ ID NO:7;

[0077] (3) CDR1 shown in SEQ ID NO:9, CDR2 shown in SEQ ID NO:10, and CDR3 shown in SEQ ID NO:11;

[0078] (4) CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO:14, and CDR3 shown in SEQ ID NO:15; and

[0079] (5) CDR1 shown in SEQ ID NO:17, CDR2 shown in SEQ ID NO:18, and CDR3 shown in SEQ ID NO:19.

[0080] In some embodiments, the BCMA-specific single-domain antibody comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with the amino acid sequence shown in any of SEQ ID NO:4, 8, 12, 16, and 20. In some embodiments, the BCMA-specific single-domain antibody comprises an amino acid sequence shown in any of SEQ ID NO:4, 8, 12, 16, and 20.

[0081] In some implementations, the single-domain antibody that specifically binds to BCMA is humanized.

[0082] In some embodiments, the BCMA-specific single-domain antibody may further include one or more additional tag sequences to facilitate purification and / or labeling. For example, the additional tag may be a His tag (such as a 6xHis tag) or an Fc tag, which facilitates the separation and purification of the peptide or helps to prolong its in vivo half-life. Those skilled in the art will understand that these additional tags do not materially affect the antibody's binding affinity.

[0083] The present invention specifically binds to the single-domain antibody of BCMA and binds to the K of BCMA. D The value can be less than approximately 1 × 10 -7 M, preferably less than about 1×10 -8 M, more preferably less than about 1×10 -9 M, more preferably less than about 1×10 -10 M.

[0084] Expression vectors and methods for preparing single-domain antibodies

[0085] In another aspect, the present invention provides isolated nucleic acid molecules encoding a single-domain antibody that specifically binds to BCMA according to the present invention. In some embodiments, the nucleotide sequence of said nucleic acid molecule is codon-optimized for a host cell for expression. In some embodiments, the nucleic acid molecule of the present invention is operatively linked to an expression regulatory element such as a promoter.

[0086] The present invention also provides an expression vector for expressing the single-domain antibody of the present invention, which comprises the nucleic acid molecule encoding the single-domain antibody of the present invention that specifically binds to BCMA.

[0087] This invention also provides host cells for preparing the single-domain antibodies of this invention, which are transformed from the nucleic acid molecules or expression vectors described above. As used herein, a "host cell" is a cell used to receive, maintain, replicate, and amplify the vector. The host cell can also be used to express nucleic acids or polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell can be a eukaryotic or prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK 293 cells.

[0088] In another aspect, the present invention provides a method for producing a single-domain antibody that specifically binds to BCMA, comprising:

[0089] (i) The host cells of the present invention are cultured under conditions suitable for expression of the nucleic acid molecules or expression vectors, and

[0090] (ii) Isolate and purify a single-domain antibody that specifically binds to BCMA expressed by the host cells.

[0091] Methods and reagents for recombinantly generating peptides, such as specific expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions, are known in the art. Similarly, protein separation and purification techniques suitable for the methods of manufacturing the BCMA-specific single-domain antibody of the present invention are well known to those skilled in the art.

[0092] However, the single-domain antibody that specifically binds to BCMA according to the present invention can also be obtained by other protein-generating methods known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.

[0093] STAR targeting BCMA

[0094] In another aspect, the present invention provides a synthetic T-cell receptor antigen receptor (STAR) that targets BCMA, comprising an antigen-binding region that specifically binds to BCMA.

[0095] In some embodiments, the synthetic T-cell receptor antigen receptor (STAR) targeting BCMA 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 BCMA.

[0096] In some embodiments, the α chain and / or the β chain further include an antigen-binding region that specifically binds to another antigen. Thus, the STAR targeting BCMA can also target the other antigen.

[0097] 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.

[0098] In some embodiments, the BCMA-targeting STAR comprises an α chain and a β chain, the α chain comprising an antigen-binding region specifically binding to BCMA and a first constant region, and the β chain comprising a second constant region. In this case, the β chain may not contain an antigen-binding region.

[0099] In some embodiments, the BCMA-targeting STAR comprises an α chain and a β chain, the α chain containing a first constant region, and the β chain containing an antigen-binding region that specifically binds to BCMA and a second constant region. In this case, the α chain may not contain an antigen-binding region.

[0100] In some embodiments, the BCMA-targeting STAR comprises an α chain and a β chain, the α chain comprising an antigen-binding region specifically binding to BCMA and a first constant region, and the β chain comprising an antigen-binding region specifically binding to another antigen and a second constant region.

[0101] In some embodiments, the BCMA-targeting STAR comprises an α chain and a β chain, the α chain comprising an antigen-binding region specifically binding to another antigen and a first constant region, and the β chain comprising an antigen-binding region specifically binding to BCMA and a second constant region.

[0102] In some embodiments, the antigen-binding region that specifically binds to BCMA comprises a single-domain antibody that specifically binds to BCMA as described in this invention. In some preferred embodiments, the antigen-binding region that specifically binds to BCMA comprises the amino acid sequence shown in SEQ ID NO:4.

[0103] The other antigen may be CD138, CD19, CD20, CD22, CD38, or GPRC5D. In some specific embodiments, the other antigen is LILRB4 or CD19.

[0104] In some embodiments, the antigen-binding region that specifically binds to another antigen comprises a single-domain antibody or a single-chain antibody (scFv) that specifically binds to the other antigen.

[0105] In some embodiments, the other antigen is LILRB4, and the antigen-binding region that specifically binds to LILRB4 comprises CDR1 shown in SEQ ID NO:38, CDR2 shown in SEQ ID NO:39, and CDR3 shown in SEQ ID NO:40. In some embodiments, the other antigen is LILRB4, and the antigen-binding region that specifically binds to LILRB4 comprises the amino acid sequence shown in SEQ ID NO:41 (single-domain antibody).

[0106] In some embodiments, the other antigen is CD19, and the antigen-binding region that specifically binds to CD19 comprises the heavy chain variable region (VH) shown in SEQ ID NO:42 and the light chain variable region (VL) shown in SEQ ID NO:43. In some embodiments, the other antigen is CD19, and the antigen-binding region that specifically binds to CD19 comprises the amino acid sequence (scFv) shown in SEQ ID NO:44.

[0107] 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:21. An exemplary natural mouse TCRα chain constant region comprises the amino acid sequence shown in SEQ ID NO:22.

[0108] In some implementations, the first constant region is a modified TCRα chain constant region.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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).

[0113] 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).

[0114] In some embodiments, the TCRα chain constant region is missing an intracellular region relative to the wild-type TCRα chain constant region, for example, by deleting amino acids 136-137.

[0115] 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.

[0116] In some specific embodiments, the modified TCRα chain constant region contains an amino acid sequence shown in one of SEQ ID NO:23-27.

[0117] 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:28. An exemplary natural mouse TCRβ chain constant region comprises the amino acid sequence shown in SEQ ID NO:29.

[0118] In some implementations, the second constant region is a modified TCRβ chain constant region.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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 lack of amino acids 167-172.

[0124] In some specific embodiments, the modified TCRβ chain constant region contains an amino acid sequence shown in one of SEQ ID NO:30-34.

[0125] In some embodiments, the first constant region comprises the amino acid sequence shown in SEQ ID NO:23, and the second constant region comprises the amino acid sequence shown in SEQ ID NO:30. In some embodiments, the first constant region comprises the amino acid sequence shown in SEQ ID NO:26, and the second constant region comprises the amino acid sequence shown in SEQ ID NO:33. In some embodiments, the first constant region comprises the amino acid sequence shown in SEQ ID NO:26, and the second constant region comprises the amino acid sequence shown in SEQ ID NO:30. In some embodiments, the first constant region comprises the amino acid sequence shown in SEQ ID NO:23, and the second constant region comprises the amino acid sequence shown in SEQ ID NO:33.

[0126] 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.

[0127] 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 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 valine (V). The α chain includes an intracellular domain of OX40 connected to the C-terminus of the constant region (e.g., directly or via a linker, such as a (G4S)n linker, where n represents an integer from 1 to 10, preferably n is 3).

[0128] 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 that is 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).

[0129] 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.

[0130] 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.

[0131] 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:35.

[0132] In some implementations, the STAR targets BMCA, and the STAR comprises...

[0133] The βα chain shown in SEQ ID NO:49 and the α chain shown in SEQ ID NO:50;

[0134] The β chain shown in SEQ ID NO:51 and the α chain shown in SEQ ID NO:52;

[0135] The β chain shown in SEQ ID NO:53 and the α chain shown in SEQ ID NO:54;

[0136] The β chain shown in SEQ ID NO:55 and the α chain shown in SEQ ID NO:56; or

[0137] The β chain shown in SEQ ID NO:57 and the α chain shown in SEQ ID NO:58.

[0138] In some implementations, the STAR targets BMCA and LILRB4, and the STAR contains...

[0139] The β chain shown in SEQ ID NO:59 and the α chain shown in SEQ ID NO:60;

[0140] The β chain shown in SEQ ID NO:61 and the α chain shown in SEQ ID NO:62;

[0141] The β chain shown in SEQ ID NO:63 and the α chain shown in SEQ ID NO:64;

[0142] The β chain shown in SEQ ID NO:65 and the α chain shown in SEQ ID NO:66; or

[0143] The β chain shown in SEQ ID NO:67 and the α chain shown in SEQ ID NO:68.

[0144] In some implementations, the STAR targets BMCA and CD19, and the STAR contains...

[0145] The β chain shown in SEQ ID NO:69 and the α chain shown in SEQ ID NO:70;

[0146] The β chain shown in SEQ ID NO:71 and the α chain shown in SEQ ID NO:72;

[0147] The β chain shown in SEQ ID NO:73 and the α chain shown in SEQ ID NO:74;

[0148] The β chain shown in SEQ ID NO:75 and the α chain shown in SEQ ID NO:76 or

[0149] The β chain shown in SEQ ID NO:77 and the α chain shown in SEQ ID NO:78.

[0150] CAR targeting BCMA

[0151] In another aspect, the present invention provides a chimeric antigen receptor (CAR) targeting BCMA, comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain comprises a single-domain antibody that specifically binds to BCMA as described in the present invention.

[0152] In some embodiments, the CAR further includes a transmembrane domain, such as a CD8α transmembrane domain or a CD28 transmembrane domain, preferably a CD8α transmembrane domain.

[0153] In some embodiments, the CAR further includes a hinge region located between the extracellular antigen-binding domain and the transmembrane domain, for example, the hinge region being the CD8α hinge region.

[0154] In some embodiments, the CAR further includes a signal transduction domain, such as a signal transduction domain that can be used for T cell activation, for example, a signal transduction domain selected from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, and CD66d. In some preferred embodiments, the CAR includes a CD3ζ signal transduction domain.

[0155] In some embodiments, the CAR further includes one or more co-stimulatory domains, such as co-stimulatory domains selected from CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, and 4-1BBL.

[0156] In some embodiments, the CAR includes, from the N-terminus to the C-terminus, the extracellular antigen-binding domain, the hinge region, the transmembrane domain, the co-stimulatory domain, and the signal transduction domain. In some embodiments, the hinge region is a CD8α hinge region, the transmembrane domain is a CD8α transmembrane domain, the signal transduction domain is a CD3ζ signal transduction domain, and the co-stimulatory domain is a 4-1BB co-stimulatory domain.

[0157] In some embodiments, the extracellular antigen-binding domain further includes an antigen-binding region that specifically binds to another antigen. Thus, the CAR can also target the other antigen. Preferably, the antigen-binding region that specifically binds to the other antigen includes a single-chain antibody (scFv) or a single-domain antibody that specifically binds to the other antigen.

[0158] In some embodiments, the other antigen is LILRB4, and the antigen-binding region that specifically binds to LILRB4 comprises CDR1 shown in SEQ ID NO:38, CDR2 shown in SEQ ID NO:39, and CDR3 shown in SEQ ID NO:40. In some embodiments, the other antigen is LILRB4, and the antigen-binding region that specifically binds to LILRB4 comprises the amino acid sequence shown in SEQ ID NO:41 (single-domain antibody).

[0159] In some embodiments, the other antigen is CD19, and the antigen-binding region that specifically binds to CD19 comprises the heavy chain variable region (VH) shown in SEQ ID NO:42 and the light chain variable region (VL) shown in SEQ ID NO:43. In some embodiments, the other antigen is CD19, and the antigen-binding region that specifically binds to CD19 comprises the amino acid sequence (scFv) shown in SEQ ID NO:44.

[0160] In some specific embodiments, the CAR contains the amino acid sequence shown in SEQ ID NO:79 or 80.

[0161] Therapeutic immune cells

[0162] In another aspect, the present invention provides an isolated therapeutic immune cell comprising (expressing) the STAR or CAR of the present invention.

[0163] In some embodiments, the immune cells are T cells. In other embodiments, the immune cells are NK cells.

[0164] In some embodiments, the therapeutic immune cells co-express the STAR or CAR of the present invention and the membrane-bound IL-15 protein (mbIL-15).

[0165] mbIL-15 or mbIL15 refers to a fusion protein formed by linking IL-15 to 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:45, 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:45. An exemplary amino acid sequence of the extracellular domain of IL-15Ra is shown in SEQ ID NO:46, 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:46. An exemplary amino acid sequence of the linker connecting the extracellular domain of IL-15Ra to IL-15 is shown in SEQ ID NO:47. An exemplary amino acid sequence of mbIL-15 is shown in SEQ ID NO:48, 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:48.

[0166] 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.

[0167] In some embodiments, the immune cells, such as T cells, described in this invention are isolated (ex vivo) immune cells, such as cells.

[0168] 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.

[0169] In some embodiments, the therapeutic immune cells, such as T cells, are therapeutic immune cells, such as T cells, that can be obtained or acquired via the expression vector of the present invention or the method of the present invention as described below.

[0170] Expression vectors and methods for preparing therapeutic immune cells

[0171] In one aspect, the present invention provides an expression vector comprising the coding sequence of the STAR or CAR of the present invention.

[0172] In some embodiments, the expression vector further comprises the coding sequence of the membrane-bound IL-15 protein (mbIL-15) of the present invention.

[0173] 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. Preferably, the promoter is an MND promoter. An exemplary nucleotide sequence of an 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.

[0174] In some implementations, the mbIL-15 can be expressed by a separate promoter.

[0175] In some embodiments, the expression vector comprises

[0176] a) A nucleotide sequence encoding a fusion polypeptide comprising the STAR α chain and the STAR β chain of the present invention linked by a self-cleaving peptide;

[0177] b) The encoding nucleotide sequence of the fusion polypeptide comprising the STAR α chain, the STAR β chain, and the mbIL-15 of the present invention linked by a self-cleaving peptide; or

[0178] c) The encoding nucleotide sequence of the fusion polypeptide of the CAR of the present invention and the mbIL-15 of the present invention linked by a self-cleaving peptide.

[0179] 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.

[0180] 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:37.

[0181] 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. In some embodiments, the fusion polypeptide may include the CAR, 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.

[0182] In some embodiments, the α chain and the β chain each contain a GM-CSF signal peptide. In some embodiments, the mbIL-15 contains an IgE signal peptide.

[0183] In another aspect, the present invention provides a method for preparing therapeutic immune cells, comprising:

[0184] Step 1) Provide initial immune cells;

[0185] Step 2) Introduce the expression vector of the present invention into the initiating immune cells; and

[0186] Step 3) Harvest the immune cells obtained in Step 2).

[0187] In some embodiments, the initiating immune cell is a T cell. In other embodiments, the initiating immune cell is an NK cell.

[0188] 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.

[0189] 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.

[0190] In some embodiments, the method described in this invention is an in vitro method.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] In some embodiments, the method further includes step y) screening for immune cells, such as T cells, expressing the STAR or CAR, or immune cells, such as T cells, co-expressing mbIL-15 and the STAR or CAR. 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.

[0195] In another aspect, the present invention provides therapeutic immune cells, such as T cells, that can be obtained or acquired through the expression vector of the present invention or the method of the present invention.

[0196] Pharmaceutical Compositions and Applications

[0197] In another aspect, the present invention provides a pharmaceutical composition comprising the single-domain antibody of the present invention, the therapeutic immune cells of the present invention, and / or the expression vector of the present invention, and a pharmaceutically acceptable vector.

[0198] In another aspect, the present invention provides the use of the single-domain antibody of the present invention, the therapeutic immune cell of the present invention, the expression vector of the present invention, and / or the pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease in a subject.

[0199] 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 single-domain antibody of the present invention, the therapeutic immune cell of the present invention, the expression vector of the present invention, and / or the pharmaceutical composition of the present invention.

[0200] In practical applications, the dosage levels of antibodies, cells, 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, without toxicity 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.

[0201] The antibodies, expression vectors, therapeutic immune cells, or pharmaceutical compositions or drugs according to the present invention can be administered in any convenient manner, including by injection, infusion, implantation, or transplantation. The antibodies, expression vectors, therapeutic immune cells, or pharmaceutical compositions described herein can be administered intravenously, intralymphaticly, intradermally, intratumorally, intramedullaryly, intramuscularly, or intraperitoneally. In one embodiment, the antibodies, expression vectors, therapeutic immune cells, or pharmaceutical compositions of the present invention are preferably administered by intravenous injection.

[0202] In embodiments of various aspects of the present invention, the disease is a BCMA-related disease, such as a disease related to abnormal BCMA expression, such as a BCMA-related cancer. The cancer is, for example, myeloma, such as multiple myeloma (MM), particularly relapsed or refractory multiple myeloma (RRMM). The disease can 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.

[0203] STAR Structure and Optimization

[0204] 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.

[0205] 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.

[0206] 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.

[0207] The inventors have previously modified the constant region of STAR to improve its performance. Specifically, this includes:

[0208] 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.

[0209] 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).

[0210] 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).

[0211] 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).

[0212] 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. These co-stimulatory molecules can be linked via adapters, such as the (G4S)3 adapter, or directly to the C-terminus of the α-chain constant region and / or the β-chain constant region without adapters. 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.

[0213] Example

[0214] The lentiviral vectors and lentiviral packaging plasmids used in the embodiments of this application were purchased from commercial companies or synthesized by commercial companies. The gene fragments used in the embodiments of this application, including signal peptides, antibody-binding regions, hinge regions, TCR constant regions, and tag proteins, were all derived from commercially synthesized companies. The embodiments of this application are merely for further description of the invention and are not intended to limit the scope of the invention.

[0215] Example 1: Screening of BCMA-targeting nanobodies

[0216] 1.1. Immunization of alpacas with human BCMA protein

[0217] Healthy alpacas were immunized with commercially available human BCMA-Fc antigen protein (manufacturer: Biointron, catalog number: B528001), using adjuvants including complete Freund's adjuvant (CFA, Sigma) and incomplete Freund's adjuvant (IFA, Sigma). The expressed and purified human BCMA protein was diluted with PBS and then mixed 1:1 with the corresponding adjuvant. The antigen and adjuvant were thoroughly mixed to form a stable emulsion. The antigen mixture was drawn into a syringe and injected subcutaneously at multiple points on the alpaca's neck, with 100-200 μL injected at each point. The specific immunization procedure for the animals is as follows:

[0218] 1) Day 1 (first immunization): Immunize alpacas with BCMA antigen (100 μg) mixed with complete Freund's adjuvant (CFA) via subcutaneous injection;

[0219] 2) Day 14 (second immunization): Immunize alpacas with BCMA antigen (100 μg) mixed with incomplete Freund's adjuvant (IFA) via subcutaneous injection;

[0220] 3) Day 28 (3rd immunization): The alpaca was immunized again by subcutaneous injection of BCMA antigen (100 μg) mixed with incomplete Freund's adjuvant (IFA);

[0221] 4) Day 42 (first serum collection): Alpaca blood samples were collected from the marginal ear vein, serum was extracted, and antibody titer was tested. The P / N value of the serum diluted 200,000 times was greater than 2.

[0222] 5) Day 42 (4th immunization): The alpaca was immunized again with a subcutaneous injection of BCMA antigen (100 μg) mixed with incomplete Freund's adjuvant (IFA) to enhance the immune effect;

[0223] 6) Day 53 (Second serum collection): Alpaca blood samples were collected from the marginal ear vein, serum was extracted, and antibody titer was tested. The P / N value of the serum diluted 200,000 times was greater than 2.

[0224] 7) Days 54, 57, and 60: Collect 30-40 mL of alpaca blood from the hind leg vein for PBMC separation.

[0225] 1.2. PBMC Separation

[0226] 1) Separate PBMCs in a biosafety cabinet, combine the blood from the anticoagulant tube into a 50mL centrifuge tube (30mL), add PBS to a total volume of 50mL, and mix gently.

[0227] 2) Take a new 50mL centrifuge tube and add Ficoll separation buffer, 15mL / tube. Spread the blood sample on the surface of the Ficoll buffer, 25mL / tube. The process should be steady to allow the Ficoll and blood to separate into layers and prevent mixing.

[0228] 3) Set the centrifuge speed to 0, maintain room temperature, centrifuge at 800g for 30 minutes. After centrifugation, remove the sample from the centrifuge. The sample layers are as follows: upper aqueous phase - white film layer - Ficoll layer - red blood cell layer, where PBMCs are in the white film layer. Aspirate the white film layer and transfer it to a new 50mL centrifuge tube.

[0229] 4) Add PBS to 50 mL in the sample tube, mix well, centrifuge at 2000 rpm at room temperature for 5 min, discard the supernatant, and resuspend the cell pellet in 5 mL of PBS.

[0230] 5) Repeat step 4) and cool down to 4℃.

[0231] 6) Resuspend the cell pellet in 5 mL of PBS, add PBS to make up to 40 mL, and count the cells.

[0232] 7) Centrifuge at 1500 rpm, 4℃ for 5 min, discard the supernatant, add 1 mL PBS, resuspend the cells, and mix well; add 20 mL Trizol, mix well, incubate at room temperature for 5 min, lyse the cells, aliquot 1 mL into RNase-free 1.5 mL EP tubes, freeze at -80℃ for RNA extraction.

[0233] 1.3. RNA extraction and reverse transcription

[0234] 1) Remove the sample from -80℃, thaw at room temperature, add 200μL of chloroform, and let stand at room temperature for 3 min. Centrifuge the sample at 12000g for 15 min at 4℃. The sample will separate into an upper aqueous phase, an intermediate layer, and an organic layer. Transfer the upper layer to a new RNase-free tube, add 1μL of glycogen and 500μL of isopropanol, and let stand at 4℃ overnight.

[0235] 2) Centrifuge the sample at 12000g for 20 min at 4℃, remove the supernatant, add 1 mL of pre-cooled 75% ethanol to wash the precipitate, centrifuge again to remove the ethanol, and air dry. Add 15 μL of RNase-free water to each tube to dissolve the RNA precipitate and perform reverse transcription.

[0236] 3) cDNA was synthesized using the Promega reverse transcription kit (20 μL system).

[0237] Step 1: Take a certain amount of template RNA and add Oligo(dT), see Table 1;

[0238] Table 1

[0239] Step 2: Place the mixture of template RNA and Oligo(dT) at 65°C for 5 minutes for pre-denaturation, and then return it to ice.

[0240] Step 3: During pre-denaturation, RT-Mix can be prepared in advance, 8 μL per tube. The components and volumes are shown in Table 2.

[0241] Table 2

[0242] Step 4: Set up the reverse transcription program, including extension and reverse transcriptase inactivation. Once the program is complete, cDNA is obtained.

[0243] 1.4. Phage Library Construction

[0244] 1) Obtain the VHH sequence by PCR

[0245] The VHH sequence was obtained through two rounds of PCR, and homologous arms of the vector were added to both ends of the sequence.

[0246] 2) First round of PCR

[0247] Step 1: Prepare the reaction system as shown in Table 3.

[0248] Table 3

[0249] Step 2: PCR conditions are shown in Table 4.

[0250] Table 4

[0251] The PCR products were subjected to gel electrophoresis, and the target band at 0.7 kb was excised and the product was recovered.

[0252] 3) Second round of PCR

[0253] Step 1: Prepare the reaction system as shown in Table 5.

[0254] Table 5

[0255] Step 2: PCR conditions are shown in Table 6.

[0256] Table 6

[0257] The PCR products were subjected to gel electrophoresis, and the target band at 400 bp was excised and the product was recovered.

[0258] 4) Vector PCR

[0259] The vector region of phagemid was obtained by PCR and used to express the VHH sequence.

[0260] Step 1: Prepare the reaction system as shown in Table 7.

[0261] Table 7

[0262] Step 2: PCR conditions are shown in Table 8.

[0263] Table 8

[0264] The PCR products were subjected to gel electrophoresis, and the 4000bp target band was excised and the product was recovered.

[0265] 5) Ligation, purification and concentration of ligation products

[0266] The VHH fragment was ligated to the phagemid vector, and the ligation product was then concentrated.

[0267] Step 1: Prepare the reaction system as shown in Table 9.

[0268] Table 9

[0269] Step 2: Incubate the above mixture at 50°C for 2 hours, then cool it on ice.

[0270] Step 3: Purify the ligation product, remove salt ions and protein components from the ligation system, and concentrate the volume to 1 / 10 of the original volume.

[0271] 1.5. Power-to-database construction

[0272] 1) Take a tube of competent E. coli cells and thaw them on ice.

[0273] 2) Take 2 μL of the ligation product or positive control and add it to the competent cells above, then gently mix. Let it stand on ice for 1-2 min, then transfer it to a pre-cooled electroporation cuvette for electroporation.

[0274] 3) Immediately after electroporation, add 1 mL of 37℃ 2YT-G medium, rinse the electroporation cup with a pipette tip, and transfer the electroporated bacterial culture to a 15 mL centrifuge tube or a 2 mL EP tube. Incubate at 37℃ until all samples have been electroporated. (2YT-G: 2×YT medium containing 2% glucose) Transfer to a 37℃ shaker and incubate at 220 rpm for 1 h.

[0275] 4) Take 5 μL of the above bacterial solution and dilute it 10... 2 -10 5 Dilute the sample with 2YT-A (2YT plate containing 100 μg / mL ampicillin) and incubate overnight at 37°C for colony counting.

[0276] 5) Inoculate the remaining bacterial culture into 2YT-AG medium, shake to the logarithmic growth phase, add helper phages for infection, and incubate at 30°C and 220 rpm for 12-16 hours. (2YT-AG: 2×YT medium containing 2% glucose and 100 μg / mL ampicillin)

[0277] 6) Collect and concentrate the phages, and determine the titer.

[0278] 1.6. Phage library antibody screening

[0279] The phage library obtained in the above steps was subjected to three rounds of antibody screening, each round including a positive selection and a negative selection. First, the phages were incubated with the antigen peptide; phages that could not bind were discarded, leaving only the phages that bound the antigen peptide. Then, the phages were incubated with BSA for negative selection, leaving only the phages that could not bind to BSA.

[0280] The phage library obtained above was subjected to three rounds of antibody screening, each round including a positive selection and a negative selection. First, the phages were incubated with the antigen peptide; phages that could not bind were discarded, leaving only the phages that bound the antigen peptide. Then, the phages were incubated with BSA for negative selection, leaving only the phages that could not bind to BSA.

[0281] 1) Coat plate. Dilute the antigen to a concentration of 2 ng / μL with PBS and add it to a 96-well plate at 100 μL / well; prepare 2% BSA with PBS and add it to the corresponding negative wells at 100 μL / well. Seal with plastic wrap and incubate overnight at 4°C.

[0282] 2) Discard the coating solution, add 200 μL of washing buffer (washing buffer: 1% Tween 20 / PBS, pH 7.4), and wash 3 times.

[0283] 3) Sealing. Add 2% BSA blocking solution to all wells, 100 μL / well, seal with plastic wrap, and incubate at 37°C for 1 h.

[0284] 4) Discard the supernatant, add 200 μL of washing solution, and wash 3 times.

[0285] 5) Add the bacteriophage to the cation wells, 1×10 12 phages / well, diluted to 100 μL, sealed with plastic wrap, and incubated at 37°C for 1 hour.

[0286] 6) Discard the supernatant, add 200 μL of washing solution, and wash 10 times.

[0287] 7) Elution-Neutralization. Add 200 μL of eluent to the sun-facing well and neutralize to pH 7-7.4.

[0288] 8) Anion selection. Add the above eluent to the anion selection well, seal with plastic wrap, incubate at 37°C for 1 hour, collect and retain the supernatant, and perform titer detection.

[0289] 9) Take a small amount of phage after one round of panning, dilute it, and spread it on a 2×YT-A plate. Incubate at 37°C overnight. The next day, count the colonies and calculate the titer. Select single clones for sequencing to analyze sequence diversity and enrichment.

[0290] 10) The remaining bacteriophages were all used for TG1 infection.

[0291] 11) M13KO7 infection. Dilute the phage, add M13KO7 to the bacterial culture, and incubate at 37°C for 30 min; replace with 2×YT-AK medium, and incubate at 30°C and 220 rpm for 14-16 h.

[0292] 12) Concentrate the phage and detect the phage titer, then proceed to the next round of screening.

[0293] The amount of coating antigen is reduced in the second and third rounds of screening, and the number of washing cycles is increased after the phage is incubated with the positive wells. Other steps are the same as those described above.

[0294] 1.7. Obtaining results by combining detection and sequencing

[0295] The phages obtained from the three rounds of screening were co-infected with TG1 with M13KO7 helper phages, plated on 2YT-AK plates, and single clones were picked for phage amplification. The phages were collected for binding detection to determine the usable phages / antibodies.

[0296] 1) Coat plate. Dilute the antigen to 1 ng / μL with coating buffer, 100 μL / well; add 2% BSA to the control negative well; seal with plastic wrap and incubate overnight at 4°C.

[0297] 2) Discard the coating solution in the plate and wash 3 times with 200-250 μL of washing solution.

[0298] 3) Add 200 μL of 2% BSA to all wells and seal at room temperature for 1 h.

[0299] 4) Discard the blocking solution in the plate, add 200 μL of washing solution, and wash once.

[0300] 5) Add 100 μL of phage to each of the positive and negative wells and incubate at 37°C for 1 h.

[0301] 6) Discard the phages in the plate, add 200 μL of washing buffer, and wash 3 times.

[0302] 7) Dilute the anti-M13-HRP antibody at 50 ng / well and incubate at room temperature for 1 hour.

[0303] 8) Discard the antibody from the plate, add 200 μL of washing buffer, and wash 5 times.

[0304] 9) Add 100 μL of TMB colorimetric solution to each well and react at room temperature until the OD value is between 2 and 3.

[0305] 10) Add stop solution to the color development system, 50 μL per well.

[0306] 11) Measure the absorbance at 450 nm using a spectrophotometer. Send the monoclonal bacterial culture corresponding to the positive well for sequencing to determine the VHH sequence.

[0307] 12) The 23 antibodies obtained were named NBCs (NBC01-NBC23).

[0308] Example 2: Performance testing of BCMA nanobody BLI antibody affinity antibody.

[0309] 2.1. BCMA Nanobody BLI Affinity Detection

[0310] Antibody affinity is determined using biomembrane interference (BLI) technology, utilizing Fortebio The detection instrument determined the antibody affinity. After immobilizing the NBCs antibodies onto the Protein A biosensor, the antibodies could bind to the Human BCMA His tag protein. The determined affinity was calculated using the Octet RED96e (ForteBio) Data Analysis 11 software. The results are shown in Table 10 and Figures 1(A)-(F). The affinity (KD value) of NBC11, NBC15, NBC16, NBC21, and NBC23 to BCMA protein were 4.27 nM, 7.29 nM, 9.50 nM, 6.14 nM, and 13.6 nM, respectively, with NBC11 showing the best affinity. The sequences of the above antibodies are shown in Table 11.

[0311] Table 10. BCMA Nanobody BLI Affinity Detection

[0312] Table 11 BCMA Nanobody Sequences

[0313] 2.2 Competitive binding of BCMA nanobody to BLI epitope

[0314] Competitive binding of antibodies is achieved via biomembrane interference (BLI) technology, utilizing Fortebio The detection was performed using advanced instrument technology. The stationary phase was BCMA protein, and the mobile phase was BCMA nanobodies (antibodies formed by the fusion of BCMA nanobodies with the Fc phase, namely NBC11-Fc, NBC15-Fc, and NBC16-Fc). In the BLI antibody competitive binding assay, the bin (classification bin) refers to the functional grouping of antibody pairs according to the competitive pattern between antibodies. As shown in Figure 2, the three detected antibodies, NBC11-Fc, NBC15-Fc, and NBC16-Fc, were all in the same bin, indicating that they recognize the same / highly overlapping epitopes, and there is complete competition between the antibodies, preventing them from binding to the antigen simultaneously.

[0315] Table 12 Results of antibody epitope inhibition rate

[0316] 2.3. BCMA Nanobody MPA Specificity Detection

[0317] The MPA developed and used by Integral Molecular is an array of over 5220 human membrane proteins (covering 94% of human membrane proteins). Each human membrane protein in the MPA has a complete structure and can be expressed in its native conformation within living cells. The MPA is an in vitro tool that enables rapid and comprehensive screening of candidate therapeutic agents for specificity. As shown in Figures 3(A)-(E), antibodies NBC11-Fc, NBC15-Fc, NBC16-Fc, NBC21-Fc, and NBC23-Fc all specifically bind to BCMA without any off-target risk.

[0318] Example 3: Single-target BCMA STAR performance verification

[0319] 3.1. Construction of BCMA STAR expression vector and lentiviral packaging

[0320] The STAR structure targeting BCMA is shown in Figure 4A. The BCMA nanobodies NBC01 to NBC23 sequences were ligated into the constant regions of the STAR molecules (TRAC(Cys-TM) and TRBC(Cys-TM)). All of these STARs contain an OX40 co-stimulatory domain that is either linked to the C-terminus of the TRAC or TRBC constant region via a linker or directly. The complete BCMA-STAR plasmid was then constructed by inserting it into a lentiviral vector using homologous recombination.

[0321] Lentix-293T cells were divided into groups of 5 × 10 5 Inoculate cells / mL into 10cm culture dishes and incubate at 37℃ with 5% CO2. Transfect when the cell density reaches approximately 80% (observed under a microscope). Mix the four plasmids (PMD2.G:PRSV-Rev:PMDlg:transfer plamid) with 500μL of serum-free DMEM in a ratio of 1:1:2:4. Mix 54μL of PEI-max with 500μL of serum-free DMEM and incubate at room temperature for 5min (PEI-Max to plasmid volume ratio is 3:1). Slowly add the PEI-max mixture to the plasmid mixture, gently pipette to mix, and incubate at room temperature for 15min. Slowly add the final mixture to the culture medium, mix thoroughly, and return to the incubator for 12-16h. Transfer to 6% FBSDMEM medium and continue culturing. Harvest virus solution at 48h and 72h.

[0322] Jurkat-C4 cells with TCR knockout were used at 1.5 × 10⁻⁶ 5 Cells were seeded at a rate of 100 μL / mL in 96-well plates with 100 μL of 1640 medium containing 10% FBS and 0.2 μL of 1000× polybrene in each well. For virus dilution, 10-fold serial dilutions were performed using complete 1640 medium. The diluted cells were added to the virus wells at a rate of 100 μL / well, mixed, centrifuged at 32°C and 1500 rpm for 90 min, and incubated at 37°C with 5% CO2. After 72 h, the infection efficiency was measured by flow cytometry. To calculate the titer, wells with an infection rate of 2-30% were selected. The formula was: Titer (TU / mL) = 1.5 × 10^4 × Positive Rate ÷ Virus Volume (μL) × 1000. The virus was then used to infect T cells to express STAR.

[0323] After thawing, frozen PBMCs were cultured in X-VIVO medium containing 10% FBS and 10 ng / mL IL-7, 10 ng / mL IL-15, and 5 ng / mL IL-21, with an initial culture density of 1×10⁻⁶. 6The virus solution was added to the culture medium at a concentration of / mL, along with the novel T-cell activation reagent T Cell TransAct conjugated with humanized CD3 and CD28 antibodies for activation. After 24 hours of activation, the virus solution was added, mixed well, and incubated in a CO2 incubator. After 48 hours of infection, X-VIVO medium containing 10% FBS and 10 ng / mL IL-7, 10 ng / mL IL-15, and 5 ng / mL IL-21 was added and the culture was transfected into wells. Subculture was performed every 1-2 days thereafter.

[0324] 3.2. BCMA STAR in vitro killing screening

[0325] Luciferase is a common substance used in cell function research. Enzyme activity is determined by adding a luciferase substrate to a system, and luciferase activity is closely related to the expression and binding strength of the target gene, as well as the number of cells. In this invention, a target cell line stably expressing luciferase is established, and the amount of luciferase is used to indicate the number of target cells, thereby indicating the cytotoxic function of functional cells.

[0326] In the experiment, the NBC01-STAR to NBC23-STAR vectors were expressed in T cells, with uninfected STAR lentivirus T cells (MOCK-T) used as a control. Raji target cells overexpressing the BCMA target (Raji-luc-BCMA) were constructed at a rate of 4 × 10⁻⁶. 5 The cells were seeded at a density of 1 mL in a 24-well plate. STAR-T cells were added to the target cells at a ratio of 0.6:1, with STAR-positive T cells and target cells respectively. 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 the target cells.

[0327] As shown in Figure 5, NBC03-STAR, NBC11-STAR, NBC14-STAR, NBC15-STAR, NBC16-STAR, NBC17-STAR, NLB20-STAR, NBC21-STAR, NBC22-STAR, and NBC23-STAR exhibit good specific killing and specific recognition of BCMA target cells, and their killing level of target cells is higher than that of other NBCs-FMC-STAR-T cells.

[0328] 3.3. BCMA STAR Cytokine Secretion Screening

[0329] Based on the above killing experiments, T cells with high killing efficiency (NBC03-STAR, NBC11-STAR, NBC14-STAR, NBC15-STAR, NBC16-STAR, NBC17-STAR, NLB20-STAR, NBC21-STAR, NBC22-STAR, and NBC23-STAR) were screened. After co-culturing T cells with target cells, the supernatant was collected. The secretion levels of IFN-γ, IL-2, and TNF-α were detected by ELISA.

[0330] 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 use the Human IL-2 Uncoated ELISA, Human TNF-α Uncoated ELISA, and Human IFN-γ Uncoated ELISA kits (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.

[0331] As shown in Figure 6, co-culturing T cells with target cells Raji-luc-BCMA significantly stimulated T cells to secrete IL-2, TNF-α, and IFNγ. The T cell cytokine concentrations or secretion levels of NBC11-STAR, NBC15-STAR, NBC16-STAR, NBC20-STAR, NBC21-STAR, NBC22-STAR, and NBC23-STAR cells were relatively high, which may be related to the higher target binding and recognition ability of their BCMA antibodies, potentially leading to a higher tumor-killing effect.

[0332] 3.4 BCMA STAR in vivo functional evaluation

[0333] 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).

[0334] To verify the in vivo cytotoxic effect of STAR-T cells and potential safety issues, a Raji-luc-BCMA tumor model overexpressing BCMA was constructed. The effects of NBC11-STAR, NBC15-STAR, NBC16-STAR, and uninfected STAR-T cells (MOCK-T) on in vivo efficacy and proliferation were investigated. Fluorescently labeled target cells were reinfused via tail vein into 6-8 week old female NPG mice at a dose of 2 × 10⁻⁶. 6 / animal, on day 6 after infusion, NBC11-STAR, NBC15-STAR, and NBC16-STAR, as well as uninfected STAR T cells (MOCK-T), were infused via tail vein, with a dose of 1.5 × 10⁹ / animal. 6 / each. Then, on days -1, 7, 10, 14, 20, and 28, tumor growth, tumor fluorescence value, weight change, and peripheral blood flow cytometry (CD3, CD8, mTCRb, BCMA protein) were detected using luciferin substrate catalytic luminescence assay.

[0335] As shown in Figures 7(A)-(C), the three types of NBC11-STAR, NBC15-STAR, and NBC16-STAR T cells all exhibited tumor-suppressive effects against BCMA+ target cells. Before day 28, NBC11-STAR T cells showed the best tumor-suppressive effect against BCMA+ target cells, and the body weight of mice using any of the three T cell types did not decrease significantly, indicating relatively good safety. Survival monitoring revealed that NBC11-STAR-T cells had relatively good survival, remaining relatively stable for the first 80 days.

[0336] Example 4: Dual-target BCMA-CD19 STAR screening

[0337] 4.1. Construction of dual-target CD19-BCMA STAR vector and viral packaging

[0338] The STAR structures targeting BCMA and CD19 are shown in Figure 4C. The BCMA nanobodies NBC01 to NBC23 sequences were co-assembled with the CD19 antibody FMC63 sequence into the constant regions of the STAR molecules (TRAC(Cys-TM) and TRBC(Cys-TM)). All of 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. Subsequently, homologous recombination was used to insert the plasmid into a lentiviral vector to construct the complete BCMA-CD19-STAR plasmid.

[0339] 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.

[0340] 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.

[0341] 4.2. Detection of dual-target CD19-BCMA STAR co-expression

[0342] After thawing frozen PBMCs, they were cultured in X-VIVO medium containing 10% FBS and 10 ng / mL IL-7, 10 ng / mL IL-15, and 5 ng / mL IL-21 at an initial density of 1 × 10⁶ / mL. Activation was then performed using T Cell TransAct, a novel T-cell activation reagent conjugated with humanized CD3 and CD28 antibodies. After 24 hours of activation, viral load was added, mixed thoroughly, and incubated in a CO₂ incubator. Forty-eight hours after infection, X-VIVO medium containing 10% FBS and 10 ng / mL IL-7, 10 ng / mL IL-15, and 5 ng / mL IL-21 was added, and the cells were transfected into wells. Subculture was performed every 1-2 days thereafter.

[0343] The above-mentioned vectors were packaged into lentiviruses and used to infect T cells. After one week of culture, the expression level of STAR cells on the cell membrane was detected by flow cytometry using anti mTCRβ antibody staining. As shown in Figure 8, the membrane expression efficiency of each structure was greater than 70%. The membrane expression rates of T cells with NBC08-FMC-STAR, NBC15-FMC-STAR, NBC16-FMC-STAR, NBC17-FMC-STAR, NBC18-FMC-STAR, NBC20-FMC-STAR, NBC22-FMC-STAR, and NBC23-FMC-STAR were slightly lower, while the membrane expression rates of other NBCs-STAR-T cells were better.

[0344] 4.3. Evaluation of the in vitro killing effect of dual-target CD19-BCMA STAR

[0345] The NBC01-FMC-STAR to NBC23-FMC-STAR vectors were expressed in T cells, with FMC-STAR and uninfected STAR T cells (MOCK-T) serving as controls. Raji-luc-CD19KO-BCMA target cells expressing only BCMA and Raji-luc target cells expressing only CD19 were separately expressed at 4 × 10⁻⁶ cells per cell line. 5The cells were seeded at a density of 1 mL in 24-well plates. NBCs-FMC-STAR-T cells were added to the target cells at a ratio of 0.6:1 (STAR-positive T cells to target cells), with a co-culture volume of 1 mL. After 24 hours of co-culture, the cell suspension was collected, and the LUC (luciferase-reporter gene assay) value was measured using a luciferase reporter gene assay kit to calculate the killing efficiency of STAR-T cells against target cells. Raji-luc-CD19KO-BCMA cells (Raji cells with CD19 knocked out and BCMA overexpressed) were used to detect the recognition and killing ability of different BCMA antibodies against the BCMA target in STAR-T cells. Raji-luc cells expressing only the CD19 target were used to detect the effect of BCMA antibody expression in STAR-T cells on the recognition and killing effect of FMC63 antibody against CD19 protein.

[0346] As shown in Figure 9(A), the in vitro killing effect of Raji-luc-CD19KO-BCMA target cells expressing only the BCMA target was as follows: STAR-T cells with dual targets NBC03-FMC, NBC05-FMC, NBC07-FMC, NBC11-FMC, NBC12-FMC, NBC13-FMC, NBC18-FMC, NBC21-FMC, NBC22-FMC, and NBC23-FMC showed higher killing levels of target cells than other NBCs-FMC-STAR-T cells. Cells; As shown in Figure 9(B), the in vitro killing effect of Raji-luc target cells expressing only CD19: STAR-T cells of NBC03-FMC, NBC05-FMC, NBC07-FMC, NBC11-FMC, NBC12-FMC, NBC14-FMC, NBC18-FMC, NBC20-FMC, NBC21-FMC, NBC22-FMC and NBC23-FMC showed higher killing levels of target cells than other NBCs-FMC-STAR-T cells.

[0347] 4.4. Dual-target CD19-BCMA STAR in vivo functional screening

[0348] NBCs-FMC-STAR-T cells with strong cytotoxic effects identified in the above-mentioned cytotoxicity assays were detected by ELISA. T cells from NBC03-FMC-STAR, NBC11-FMC-STAR, NBC14-FMC-STAR, NBC15-FMC-STAR, NBC16-FMC-STAR, NBC17-FMC-STAR, NLB20-FMC-STAR, NBC21-FMC-STAR, NBC22-FMC-STAR, and NBC23-FMC-STAR were used for further screening to determine NBCs-FMC-STAR cells with even better in vivo efficacy against tumor cells. The screening and validation of STAR-T cells in vivo was conducted to verify their cytotoxic effect and potential safety issues.

[0349] A Raji-luc-CD19KO-BCMA target cell tumor model expressing only BCMA was constructed to investigate the effects of NBCs-FMC-STAR-T cells and uninfected STAR-T cells (MOCK-T) on in vivo drug efficacy and expansion. Fluorescently labeled target cells were reinfused via tail vein into 6-8 week old female NPG mice at a dose of 2 × 10⁻⁶. 6 / animal, on day 6 after infusion, the T-cell dose group receiving STAR via tail vein infusion was 2×10 6 / each. Then, on days -1, 6, 9, 15, 20, and 35, tumor growth, tumor fluorescence values, weight changes, and peripheral blood flow cytometry (CD3, CD8, mTCRb) were detected using a luciferin substrate catalytic luminescence method.

[0350] As shown in Figures 10(A)-(C), the dual-target NBC11-FMC-STAR-T cells exhibited the strongest inhibitory effect on BCMA single-positive tumor cells (Raji-luc-CD19KO-BCMA) in mice. NBC15-FMC-STAR-T and NBC16-FMC-STAR-T cells showed the next strongest tumor-inhibiting effects, while other NBCs-FMC-STAR-T cells showed some inhibitory activity against target cell tumors. In Experiment 1, the NBC11-FMC-STAR, NBC15-FMC-STAR, and NBC16-FMC-STAR T cells screened in the in vivo experiments demonstrated significant inhibitory effects on BCMA single-positive tumor cells (Raji-luc-CD19KO-BCMA target cells) in vivo.

[0351] Example 5: Dual-target BCMA-LILRB4 STAR screening

[0352] 5.1. Construction of the dual-target LILRB4-BCMA STAR vector and viral packaging

[0353] The STAR structures targeting BCMA and LILRB4 are shown in Figure 4B. The BCMA nanobody sequences NBC01 to NBC23 and the LILRB4 nanobody sequence NLB14 (amino acid sequence shown in SEQ ID NO:41; its amino acid sequences are shown in CDR1-3 of SEQ ID NO:38-40) were co-assembled into the constant regions of the STAR molecules (TRAC(Cys-TM) and TRBC(Cys-TM)). All of these STARs contain an OX40 co-stimulatory domain linked to the C-terminus of the TRAC or TRBC constant region via a linker or directly. Subsequently, homologous recombination was used to insert the plasmid into a lentiviral vector to construct the complete BCMA-LILRB4-STAR plasmid. See Example 3 for specific methods.

[0354] Lentix-293T cells were divided into groups of 5 × 10 5 Inoculate cells / mL into 10cm culture dishes and incubate at 37℃ with 5% CO2. Transfect when the cell density reaches approximately 80% (observed under a microscope). Mix the four plasmids (PMD2.G:PRSV-Rev:PMDlg:transfer plamid) with 500μL of serum-free DMEM in a ratio of 1:1:2:4. Mix 54μL of PEI-max with 500μL of serum-free DMEM and incubate at room temperature for 5min (PEI-Max to plasmid volume ratio is 3:1). Slowly add the PEI-max mixture to the plasmid mixture, gently pipette to mix, and incubate at room temperature for 15min. Slowly add the final mixture to the culture medium, mix thoroughly, and return to the incubator for 12-16h. Transfer to 6% FBSDMEM medium and continue culturing. Harvest virus solution at 48h and 72h.

[0355] Jurkat-C4 cells with TCR knockout were used at 1.5 × 10⁻⁶ 5 Cells were seeded at a rate of 100 μL / mL in 96-well plates with 100 μL of 1640 medium containing 10% FBS and 0.2 μL of 1000× polybrene in each well. For virus dilution, 10-fold serial dilutions were performed using complete 1640 medium. The diluted cells were added to the virus wells at a rate of 100 μL / well, mixed, centrifuged at 32°C and 1500 rpm for 90 min, and incubated at 37°C with 5% CO2. After 72 h, the infection efficiency was measured by flow cytometry. When calculating the titer, wells with an infection rate of 2-30% were selected. The calculation formula is: Titer (TU / mL) = 1.5 × 10^4 × Positive Rate ÷ Virus Volume (μL) × 1000. The above virus was then used to infect T cells to express STAR.

[0356] 5.2. Detection of dual-target LILRB4-BCMA STAR co-expression

[0357] After thawing frozen PBMCs, they were cultured in X-VIVO medium containing 10% FBS and 10 ng / mL IL-7, 10 ng / mL IL-15, and 5 ng / mL IL-21 at an initial density of 1 × 10⁶ / mL. Activation was then performed using T Cell TransAct, a novel T-cell activation reagent conjugated with humanized CD3 and CD28 antibodies. After 24 hours of activation, viral load was added, mixed thoroughly, and incubated in a CO₂ incubator. Forty-eight hours after infection, X-VIVO medium containing 10% FBS and 10 ng / mL IL-7, 10 ng / mL IL-15, and 5 ng / mL IL-21 was added, and the cells were transfected into wells. Subculture was performed every 1-2 days thereafter.

[0358] The above vectors were packaged into lentiviruses and used to infect T cells. After one week of culture, the expression levels of STAR and BCMA on the cells were detected by flow cytometry. BCMA protein antibody staining detected the expression of BCMA antibody in the STAR structure on the cell membrane. RFP was used to detect the positive rate of STAR vector infection. As shown in Figure 11, the T cell proteins of NBC08-NLB14STAR, NBC10-NLB14 STAR, and NBC17-NLB14 STAR were weakly bound, while NBC18-NLB14 STAR-T cells showed almost no binding to BCMA protein.

[0359] 5.3. Dual-target LILRB4-BCMA STAR in vitro killing screening

[0360] Luciferase is a common substance used in cell function research. Enzyme activity is determined by adding a luciferase substrate to a system, and luciferase activity is closely related to the expression and binding strength of the target gene, as well as the number of cells. In this invention, a target cell line stably expressing luciferase is established, and the amount of luciferase is used to indicate the number of target cells, thereby indicating the cytotoxic function of functional cells.

[0361] NCI-H929-luc-LILRB4KO cells were constructed to verify the killing effect of NBC23s-NLB14-STAR-T cells on tumor cells expressing BCMA. The NBC01-NLB14-STAR to NBC23-NLB14-STAR vector was expressed in T cells, with NLB14-STAR and uninfected STAR T cells (MOCK-T) used as controls. NCI-luc-H929-LILRB4KO target cells expressing only BCMA were introduced at a rate of 4 × 10⁻⁶ cells / cell. 5The cells were seeded at a density of 1 mL in a 24-well plate. The NBCs-NLB14-STAR-T cells were added to the target cells at a ratio of 0.5:1 (STAR ​​positive T cells to target cells). 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 the target cells. As shown in Figure 12(A), the killing efficiency of NBCs-NLB14-STAR-T cells against the target cell NCI-H929-LILRB4KO can be observed: the STAR-T cells of dual-target NBC02-NLB14, NBC03-NLB14, NBC04-NLB14, NBC05-NLB14, NBC11-NLB14, NBC12-NLB14, NBC13-NLB14, NBC16-NLB14, NBC19-NLB14, NBC21-NLB14, and NBC23-NLB14 have a stronger killing efficiency than other NBCs-NLB14-STAR-T cells.

[0362] In addition, OCI-AML3-luc cells expressing only the LILRB4 target were selected to verify the killing level of NBC23s-NLB14-STAR-T cells against tumor cells expressing the LILRB4 target. The NBC01-NLB14-STAR to NBC23-NLB14-STAR vectors were expressed in T cells, and NLB14-STAR and uninfected STAR T cells (MOCK-T) were used as controls. OCI-AML3-luc target cells expressing only the LILRB4 target were seeded in 24-well plates at a density of 4E5 / well. STAR-T cells were added to the target cells at a ratio of 0.5:1 (STAR-positive T cells to target cells), 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. As shown in Figure 12(B), the killing efficiency of target cells OCI-AML3 can be observed: the killing efficiency of STAR-T cells targeting dual-target NBC01-NLB14, NBC02-NLB14, NBC05-NLB14, NBC07-NLB14, NBC09-NLB14, NBC11-NLB14, NBC12-NLB14, NBC14-NLB14, NBC16-NLB14, NBC17-NLB14, NBC18-NLB14, NBC19-NLB14, NBC21-NLB14, and NBC23-NLB14 is stronger than that of other NBCs-NLB14-STAR-T cells.

[0363] 5.4. Detection of Dual-Target LILRB4-BCMA STAR Cytokine Secretion

[0364] Following the above experiments, NBCs-NLB14-STAR-T cells with strong cytotoxic effects were tested. After co-culturing T cells with target cells, the supernatant was collected. The secretion levels of IFN-γ, IL-2, and TNF-α were detected by ELISA.

[0365] 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 use the Human IL-2 Uncoated ELISA, Human TNF-α Uncoated ELISA, and Human IFN-γ Uncoated ELISA kits (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.

[0366] As shown in Figure 13(A), co-culturing T cells with target cells NCI-H929-luc-LILRB4KO significantly stimulated T cells to secrete IFN-γ, IL-2, and TNF-α. The NBC-NLB14-STAR cells showed the best cytokine secretion concentrations. The cytokine secretion levels of T cells from NBC01-NLB14-STAR, NBC02-NLB14-STAR, NBC11-NLB14-STAR, NBC19-NLB14-STAR, NBC21-NLB14-STAR, and NBC23-NLB14-STAR were higher than those of other STAR-T cells. As shown in Figure 13(B), co-culturing T cells with target cells OCI-AML3-luc significantly stimulated T cells to secrete IFN-γ, IL-2, and TNF-α. The NBC-NLB14-STAR-T cells showed the highest cytokine secretion levels. The cytokine secretion levels of T cells targeting dual-target cells NBC01-NLB14-STAR, NBC02-NLB14-STAR, NBC05-NLB14-STAR, NBC11-NLB14-STAR, NBC12-NLB14-STAR, NBC19-NLB14-STAR, NBC21-NLB14-STAR, and NBC23-NLB14-STAR were higher than those of other STAR-T cells.

[0367] 5.5. In vivo functional validation of dual-target LILRB4-BCMA STAR

[0368] Based on the above cytokine secretion assay, NBC01-NLB14-STAR, NBC02-NLB14-STAR, NBC11-NLB14-STAR, NBC19-NLB14-STAR, NBC21-NLB14-STAR, and NBC23-NLB14-STAR were identified. The in vivo inhibitory effect of these STAR-T cells in a BCMA-positive tumor cell transplantation mouse model was further verified.

[0369] Raji-luc-CD19KO-BCMA target cells were constructed and subcutaneously injected into 6-8 week old female NPG mice at a dose of 2 × 10⁻⁶. 6 / animal, on day 6 after infusion, NBC01-NLB14-STAR, NBC02-NLB14-STAR, NBC11-NLB14-STAR, NBC19-NLB14-STAR, NBC21-NLB14-STAR, NBC23-NLB14-STAR, and uninfected STAR T cells (MOCK-T) were selected, with a dose of 2×10 6 / mouse. Tumor growth, tumor fluorescence value, and weight changes were detected by luciferin substrate catalytic luminescence assay 1 day before infusion and on days 4, 7, 14, and 22 after infusion to evaluate the in vivo killing effect and potential safety issues in a multiple myeloma model. Results are shown in Figures 14(A)-(C). NBC11-NLB14-STAR-T, NBC21-NLB14-STAR-T, and NBC23-NLB14-STAR-T T cells all showed good tumor suppression effects against Raji-luc-CD19KO-BCMA target cells. Among them, NBC11-NLB14-STAR-T cells showed slightly better tumor suppression effects on target cells compared to NBC21-NLB14-STAR-T and NBC23-NLB14-STAR-T cells. Furthermore, the weight of mice using any of the three T cell types did not decrease significantly, indicating relatively good safety.

[0370] 5.6. Evaluation of the killing effect of dual-target LILRB4-BCMA STAR on MM cell lines

[0371] NBC11-NLB14-STAR, NBC15-NLB14-STAR, NBC16-NLB14-STAR, NBC21-NLB14-STAR, NBC23-NLB14-STAR, NLB4-NLB14-STAR, and MOCK-T vectors were expressed in T cells. Four target cell lines expressing BCMA and LILRB4 targets—OPM2-luc, U266B1(CFSE)-luc, MM1S-luc, and RPMI-8226-luc—were distributed at a ratio of 4 × 10⁻⁶ cells / cells. 5 The cells were seeded at a density of 1 / 2 well in a 24-well plate. NBCs-NLB14-STAR-T cells were added to the target cells at a ratio of 1:1 and 0.3:1 to STAR-positive T cells and co-cultured for 24 h. The luminescence value of LUC was detected using a luciferase reporter gene assay kit, and the killing efficiency of STAR-T cells against the target cells was calculated.

[0372] The results in Figure 15(A) indicate that, under the effector-to-target ratio of 1:1, the four types of NBCs-NLB14 STAR cells showed comparable killing levels against OPM2-luc target cells. Under the effector-to-target ratio of 0.3:1, NBC11-NLB14-STAR-T cells showed better killing levels against OPM2-luc target cells. The killing levels of NBC15-NLB14 STAR-T cells were comparable to those of NBC16-NLB14 STAR-T cells and higher than those of the other NBCs-NLB14 STAR-T cells.

[0373] The results in Figure 15(B) indicate that, against U266B1-luc target cells, under an effector-to-target ratio of 1:1, NBC11-NLB14-STAR-T cells significantly increased the killing level of target cells compared to other NBCs-NLB14 STAR-T cells. The order of killing levels was: NBC11-NLB14-STAR-T > NBC16-NLB14-STAR-T > NBC23-NLB14-STAR-T > NBC15-NLB14-STAR-T > NBC21-NLB14-STAR-T. Under an effector-to-target ratio of 0.3:1, NBC16-NLB14-STAR-T cells showed a slightly higher killing level, while other NBCs-NLB14 STAR-T cells showed comparable killing levels.

[0374] The results in Figure 15(C) indicate that, against MM.1S-luc target cells, under an effector-to-target ratio of 1:1, NBC11-NLB14-STAR-T, NBC21-NLB14-STAR-T, and NBC23-NLB14-STAR-T exhibited slightly higher cell-killing levels than NBC15-NLB14-STAR-T and NBC16-NLB14-STAR-T. Under an effector-to-target ratio of 0.3:1, NBC11-NLB14-STAR-T and NBC16-NLB14-STAR-T exhibited slightly higher cell-killing levels. The order of cell-killing levels was: NBC11-NLB14-STAR-T > NBC16-NLB14-STAR-T > NBC23-NLB14-STAR-T > NBC15-NLB14-STAR-T > NBC21-NLB14-STAR-T.

[0375] Figure 15(D) shows that, against RPMI8226-luc target cells, at an effector-to-target ratio of 1:1, NBC11-NLB14-STAR-T showed a slightly higher killing level, while other NBCs-NLB14 STAR-Ts were roughly equivalent; at an effector-to-target ratio of 0.3:1, NBC11-NLB14 showed a significantly higher killing level than other NBCs-NLB14 STAR-Ts. Example 6: Co-expression of mbIL-15 by dual-target BCMA-LILRB4 STAR.

[0376] 6.1. Construction of vectors co-expressing mbIL-15 and BCMA-LILRB4 STAR and viral packaging

[0377] The STAR structure of BCMA-LILRB4 co-expressing mbIL-15 is shown in Figure 4D. The sequences of BCMA nanobodies NBC11, NBC15, NBC16, NBC21, and NBC23 were co-assembled with the LILRB4 antibody NLB14 sequence into the constant regions of the STAR molecules (TRAC(Cys-TM) and TRBC(Cys-TM)). All of 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. Subsequently, homologous recombination was used to insert the plasmid into a lentiviral vector to construct the complete BCMA-LILRB4-STAR-mbIL-15 plasmid.

[0378] 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.

[0379] 6.2. Expression and proliferation detection of LILRB4-BCMA STAR cells co-expressing mbIL-15 on the membrane.

[0380] 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 Cultured at a density of / mL, the cells were infected with the virus at an MOI of 4 after 24 hours, and passaged every other day. On the sixth day post-infection, the STAR infection efficiency was analyzed by detecting the mTCRβ ratio using flow cytometry after staining with BV421-anti mTCRβ antibody and APC-antiIL-15Ra antibody. As shown in Figure 16, NBC11-NLB14 STAR-mbIL-15, NBC15-NLB14 STAR-mbIL-15, NBC16-NLB14 STAR-mbIL-15, NBC21-NLB14 STAR-mbIL-15, and NBC23-NLB14 STAR-mbIL-15 all showed good membrane loading efficiency, and mbIL-15 showed high co-expression efficiency.

[0381] 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-incubation of T cells with target cells, the change in the number of positive T cells was detected by flow cytometry. Changes in T cell proliferation: Fold rate = Number of positive T cells at the detection time point / Initial amount of positive T cells added.

[0382] NBC11-NLB14-STAR-T-mbIL-15 cells with different infection efficiencies M1 (MOI = 1.5) and M2 (MOI = 4) were initially co-cultured with NCI-H929 target cells at an effector-to-target ratio of 0.8:1. NBC11-NLB14-STAR-T cells 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 number of NCI-H929 target cells was 1 × 10⁻⁶ cells. 5 Cells and samples at each time point were incubated independently, and the remaining co-incubated samples were replaced with new medium and target cells every 4 days. Cells used for flow cytometry analysis were stained with EF506-Live / Die, FITC-anti-human CD3, and APC-anti-mouse TCR antibodies. A specified volume of cells was collected and recorded during flow cytometry analysis, and the number and proportion of T cells in the system were calculated. As shown in Figures 17(A)-(C), the fold increase curves of absolute T cell and STAR cell numbers 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.

[0383] 6.3. Validation of the continuous kill advantage of NBC11-NLB14 STAR co-expressing mbIL-15

[0384] The NBC11-NLB14-STAR and NBC11-NLB14-mbIL-15-STAR vectors were expressed in T cells, with uninfected STAR T cells (MOCK-T) used as a control. NCI-H929-luc target cells expressing BCMA and LILRB4 targets were seeded at a ratio of 4E5 cells / well 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, 4E5 NCI-H929-luc target cells were added to each well for continued co-culturing, with target cell killing assessed every 48 hours until no further target cell killing occurred. The experiment was then terminated. The results are shown in Figure 18. The continuous killing efficiency of NBC11-NLB14 STAR-mbIL-15 was higher than that of NBC11-NLB14 STAR.

[0385] 6.4. In vivo efficacy of mbIL-15 co-expressing NBC11-NLB14 STAR mixed tumor model

[0386] Experiment 1: Sequence selection for the H929-luc-LILRB4KO model

[0387] A mouse tumor model of H929-luc-LILRB4KO target cells was constructed. STAR groups (NBC11-NLB14-STAR, NBC21-NLB14-STAR, NBC23-NLB14-STAR, NBC11-NLB14-mbIL-15-STAR, NBC21-NLB14-mbIL-15-STAR, and NBC23-NLB14-mbIL-15-STAR) were expressed in T cells, with uninfected STAR T cells (MOCK-T) used as a control. The above H929-luc-LILRB4KO target cells were subcutaneously inoculated into 6-8 week old female NPG mice at a dose of 5E6 per mouse. On day 6 post-inoculation, control MOCK-T cells, simultaneously infused with the above STAR-T groups, were infused via tail vein at a dose of 3E6 per mouse. After T-cell infusion, tumor growth was detected once or twice a week using a luciferin substrate-catalyzed luminescence method, and T-cell proliferation in mice was monitored by peripheral blood flow cytometry.

[0388] As shown in Figures 19(A)-(D), both BCMA-LILRB4 STAR-T cells and T cells co-expressing mbIL-15 with BCMA-LILRB4 STAR showed good tumor growth inhibition effects, and the tumor inhibition effects were basically equivalent.

[0389] Experiment 2: Mixture Model

[0390] 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.

[0391] The aforementioned mixed target cells were subcutaneously inoculated into 6-8 week old female NPG mice at a dose of 5E6 per mouse. On day 6 post-inoculation, NBC11-NLB14-START-mbIL15 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:81) T cells (B2, BCMA antibody from Legend Biotech's Cidarkind) and NLB14 antibody, recognizing both BCMA and LILRB4, as well as a MOCK-T cell control group, all at a dose of 1.5E6 per mouse. Starting on day 5 post-infusion, tumor growth was detected using a luciferase-based chemiluminescence assay, and T cell expansion in mice was monitored by peripheral blood flow cytometry.

[0392] As shown in Figures 20(A)-(C), the dual-target BCMA-LILRB4 STAR-T derived from the antibody of this invention showed better tumor growth inhibition compared to the single-target STAR-T control group (BCMA STAR-T, LILRB4 STAR-T) and the dual-target B2-NLB14 STAR-T control. In terms of in vivo T cell expansion levels, T cells expressing NBC11-NLB14 STAR-mbIL-15 also exhibited higher expansion levels and persistence in vivo.

[0393] Example 7: Dual-target BCMA-LILRB4 CAR

[0394] 7.1 Dual-target BCMA-LILRB4 CAR

[0395] 1) The structures shown in the table below are for constructing CAR structures targeting BCMA and LILRB4. The BCMA nanobody NBC11 sequence and the LILRB4 antibody NBL14 sequence were co-assembled into the constant region of the CAR molecule and inserted into a lentiviral vector using homologous recombination to construct a complete BCMA-LILRB4-CAR lentiviral plasmid.

[0396] 2) Packaging viruses

[0397] 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.

[0398] 3) Virus titer measurement

[0399] 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 Add virus cells / mL to the wells, centrifuge at 32℃, 1500 rpm for 90 min, incubate in an incubator, and measure the infection efficiency by flow cytometry after 72 h. Select wells with an infection rate of 2-30% for titer calculation. Titrate (TU / mL) = 1.5 × 10⁴ × positive rate ÷ virus volume (μL) × 1000.

[0400] 7.2 Detection of dual-target BCMA-LILRB4 CAR co-expression

[0401] 1) Isolation, activation and infection of human primary T cells

[0402] Primary T cells were obtained using the Ficoll method and cultured in X-VIVO medium containing 10% FBS and 100 IU / mL IL-2 at an initial density of 1×10⁶ / mL. Activation was then performed in wells pre-coated with CD3, CD28, and Fibronectin. After 24 hours of activation, virus solution was added, and the cells were centrifuged at 1500 rpm for 90 minutes and then incubated in a CO₂ incubator. 24 hours after infection, X-VIVO medium containing 10% FBS and 100 IU / mL IL-2 was added, and the cells were transfected into wells. Subculture was performed every 1-2 days thereafter.

[0403] 2) Detection of CAR infection efficiency and co-expression efficiency under different construction strategies

[0404] The above-mentioned vector was packaged into lentivirus and used to infect T cells. After one week of culture, the expression level of CAR on the cell membrane was detected by flow cytometry using anti mTCRβ antibody staining, as shown in Figure 21. The infection efficiency of NBC11-(G4S)1-NLB14-41BB CAR and NLB14-(G4S)1-NBC11-41BB CAR is shown in the table below.

[0405] 7.3 Validation of the killing advantage of dual-target BCMA-LILRB4 CAR

[0406] Luciferase is a common substance used in cell function research. Enzyme activity is determined by adding a luciferase substrate to a system, and luciferase activity is closely related to the expression and binding strength of the target gene, as well as the number of cells. In this invention, a target cell line stably expressing luciferase is established, and the amount of luciferase is used to indicate the number of target cells, thereby indicating the cytotoxic function of functional cells.

[0407] 1) Evaluation of in vitro killing effect of dual-target BCMA-LILRB4 CAR

[0408] The NBC11-(G4S)1-NLB14-41BB CAR and NLB14-(G4S)1-NBC11-41BB CAR vectors were expressed in T cells. NCI-H929-LUC target cells expressing BCMA and LILRB4 targets were seeded in 24-well plates at a density of 4E5 cells / well. The CAR-T cells were co-cultured with target cells at ratios of 1:1 and 0.3:1. 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 CAR-T cells against target cells. As shown in Figure 22(A), it can be observed that the killing level of NLB14-(G4S)1-NBC11-41BB CAR against NCI-H929-LUC target cells was higher than that of NBC11-(G4S)1-NLB14-41BB CAR.

[0409] The NLB14-(G4S)1-NBC11-41BB CAR vector was expressed in T cells. NCI-H929-luc target cells expressing BCMA and LILRB4 targets were seeded at a ratio of 4E5 cells / well in 24-well plates. CAR-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, 4E5 NCI-H929-luc target cells were added to each well for continued co-culturing, and target cell killing was assessed every 48 hours until no further target cell killing occurred. The experiment was then terminated. The results are shown in Figure 22(B), indicating that the NLB14-(G4S)1-NBC11-41BB CAR showed good continuous killing efficiency.

[0410] This invention relates to partial sequence information:

Claims

1. A single-domain antibody that specifically binds to BCMA, comprising CDR1, CDR2 and CDR3 from any one of SEQ ID NO:4, 8, 12, 16 and 20.

2. The single-domain antibody according to claim 1, comprising a subset of CDR1, CDR2, and CDR3: (1) CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3; (2) CDR1 shown in SEQ ID NO:5, CDR2 shown in SEQ ID NO:6, and CDR3 shown in SEQ ID NO:7; (3) CDR1 shown in SEQ ID NO:9, CDR2 shown in SEQ ID NO:10, and CDR3 shown in SEQ ID NO:11; (4) CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO:14, and CDR3 shown in SEQ ID NO:15; and (5) CDR1 shown in SEQ ID NO:17, CDR2 shown in SEQ ID NO:18, and CDR3 shown in SEQ ID NO:

19.

3. The single-domain antibody according to claim 1 or 2, comprising an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% sequence identity with the amino acid sequence shown in any of SEQ ID NO:4, 8, 12, 16, and 20, preferably comprising the amino acid sequence shown in any of SEQ ID NO:4, 8, 12, 16, and 20.

4. The single-domain antibody according to any one of claims 1-3, wherein the K+ binding to BCMA D The value is less than approximately 1 × 10 - 7 M, preferably less than about 1×10 -8 M, more preferably less than about 1×10 -9 M, more preferably less than about 1×10 -10 M.

5. An isolated nucleic acid molecule encoding a single-domain antibody that specifically binds to BCMA according to any one of claims 1-4.

6. An expression vector comprising the nucleic acid molecule of claim 5, said nucleic acid molecule being operatively linked to an expression regulatory element such as a promoter.

7. A host cell transformed by the nucleic acid molecule of claim 5 or the expression vector of claim 6.

8. A method for generating a single-domain antibody that specifically binds to BCMA, comprising: (i) Culturing the host cells of claim 7 under conditions suitable for expression of the nucleic acid molecule or expression vector, and (ii) Isolate and purify a single-domain antibody that specifically binds to BCMA expressed by the host cell.

9. A synthetic T-cell receptor antigen receptor (STAR) that targets BCMA, comprising an antigen-binding region that specifically binds to BCMA.

10. The STAR of claim 9, comprising 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 BCMA.

11. The STAR according to claim 9 or 10, wherein i) The BCMA-targeting STAR comprises an α chain and a β chain, wherein the α chain comprises an antigen-binding region specifically binding to BCMA and a first constant region, and the β chain comprises a second constant region; or ii) The STAR targeting BCMA comprises an α chain and a β chain, the α chain comprising a first constant region, and the β chain comprising an antigen-binding region specifically binding to BCMA and a second constant region.

12. The STAR according to any one of claims 9-11, wherein the antigen-binding region that specifically binds to BCMA comprises a single-domain antibody that specifically binds to BCMA according to any one of claims 1-4.

13. The STAR according to any one of claims 9-12, wherein the α chain and / or the β chain further comprises an antigen-binding region that specifically binds to another antigen, for example, the antigen-binding region that specifically binds to another antigen comprises a single-domain antibody or a single-chain antibody (scFv) that specifically binds to another antigen.

14. The STAR according to claim 13, wherein i) The STAR targeting BCMA comprises an α chain and a β chain, wherein the α chain comprises an antigen-binding region specifically binding to BCMA and a first constant region, and the β chain comprises an antigen-binding region specifically binding to another antigen and a second constant region; or ii) The STAR targeting BCMA comprises an α chain and a β chain, the α chain comprising an antigen-binding region specifically binding to another antigen and a first constant region, and the β chain comprising an antigen-binding region specifically binding to BCMA and a second constant region.

15. The STAR according to claim 13 or 14, wherein the other antigen is LILRB4 or CD19.

16. The STAR according to claim 15, wherein the other antigen is LILRB4, and the antigen-binding region specifically binding to LILRB4 comprises CDR1 shown in SEQ ID NO:38, CDR2 shown in SEQ ID NO:39, and CDR3 shown in SEQ ID NO:40, preferably, the antigen-binding region specifically binding to LILRB4 comprises the amino acid sequence shown in SEQ ID NO:

41.

17. The STAR according to claim 15, wherein the other antigen is CD19, and the antigen-binding region specifically binding to CD19 comprises the heavy chain variable region (VH) shown in SEQ ID NO:42 and the light chain variable region (VL) shown in SEQ ID NO:43, preferably, the antigen-binding region specifically binding to CD19 comprises the amino acid sequence shown in SEQ ID NO:

44.

18. The STAR according to any one of claims 1-17, 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.

19. The STAR of claim 18, 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.

20. The STAR according to claim 18 or 19, 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.

21. The STAR according to any one of claims 18-20, 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.

22. The STAR according to any one of claims 18-21, 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).

23. The STAR according to any one of claims 18-22, 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.

24. The STAR according to any one of claims 18-23, wherein the intracellular region of the TCRα chain constant region is missing relative to the wild-type TCRα chain constant region, for example, the 136th-137th amino acid is missing.

25. The STAR according to any one of claims 18-24, wherein 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 lack of amino acids 136-137.

26. The STAR according to any one of claims 18-25, wherein the modified TCRα chain constant region comprises the amino acid sequence shown in one of SEQ ID NO:23-27.

27. The STAR according to any one of claims 1-26, 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.

28. The STAR of claim 27, 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.

29. The STAR according to claim 27 or 28, 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.

30. The STAR according to any one of claims 27-29, 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.

31. The STAR according to any one of claims 27-30, 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 of the constant region is missing amino acids 167-172.

32. The STAR according to any one of claims 27-31, wherein 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 lack of amino acids 167-172.

33. The STAR according to any one of claims 9-32, wherein the modified TCRβ chain constant region comprises the amino acid sequence shown in one of SEQ ID NO:30-34.

34. The STAR according to any one of claims 9-33, wherein i) The first constant region contains the amino acid sequence shown in SEQ ID NO:23, and the second constant region contains the amino acid sequence shown in SEQ ID NO:30; ii) The first constant region contains the amino acid sequence shown in SEQ ID NO:26, and the second constant region contains the amino acid sequence shown in SEQ ID NO:33; iii) The first constant region contains the amino acid sequence shown in SEQ ID NO:26, and the second constant region contains the amino acid sequence shown in SEQ ID NO:30; or iv) The first constant region contains the amino acid sequence shown in SEQ ID NO:23, and the second constant region contains the amino acid sequence shown in SEQ ID NO:

33.

35. The STAR according to any one of claims 9-34, 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:

35.

36. The STAR of claim 35, wherein the exogenous intracellular functional domain is directly or via a linker connected to the C-terminus of the constant region of the α-chain and / or β-chain. Preferably, the connector is a (G4S)n connector, where n represents an integer from 1 to 10, and preferably, n is 3.

37. The STAR according to claim 36, wherein 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 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 valine (V). The α chain contains 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). 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).

38. A synthetic T-cell receptor antigen receptor (STAR) targeting BCMA, wherein the STAR comprises The β chain shown in SEQ ID NO:49 and the α chain shown in SEQ ID NO:50; The β chain shown in SEQ ID NO:51 and the α chain shown in SEQ ID NO:52; The β chain shown in SEQ ID NO:53 and the α chain shown in SEQ ID NO:54; The β chain shown in SEQ ID NO:55 and the α chain shown in SEQ ID NO:56; The β chain shown in SEQ ID NO:57 and the α chain shown in SEQ ID NO:58; The β chain shown in SEQ ID NO:59 and the α chain shown in SEQ ID NO:60; The β chain shown in SEQ ID NO:61 and the α chain shown in SEQ ID NO:62; The β chain shown in SEQ ID NO:63 and the α chain shown in SEQ ID NO:64; The β chain shown in SEQ ID NO:65 and the α chain shown in SEQ ID NO:66; The β chain shown in SEQ ID NO:67 and the α chain shown in SEQ ID NO:68; The β chain shown in SEQ ID NO:69 and the α chain shown in SEQ ID NO:70; The β chain shown in SEQ ID NO:71 and the α chain shown in SEQ ID NO:72; The β chain shown in SEQ ID NO:73 and the α chain shown in SEQ ID NO:74; The β chain shown in SEQ ID NO:75 and the α chain shown in SEQ ID NO:76; or The β chain shown in SEQ ID NO:77 and the α chain shown in SEQ ID NO:

78.

39. A chimeric antigen receptor (CAR) targeting BCMA, comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain comprises a single-domain antibody that specifically binds to BCMA as described in this invention.

40. The CAR of claim 39, wherein the CAR comprises, from the N-terminus to the C-terminus, the extracellular antigen-binding domain, the hinge region, the transmembrane region, the co-stimulatory domain, and the signal transduction domain.

41. The CAR of claim 39 or 40, wherein the extracellular antigen-binding domain further comprises an antigen-binding region that specifically binds to another antigen, preferably, the antigen-binding region that specifically binds to the other antigen comprises a single-chain antibody (scFv) or a single-domain antibody that specifically binds to the other antigen.

42. The CAR of claim 41, wherein the other antigen is LILRB4, and the antigen-binding region specifically binding to LILRB4 comprises CDR1 shown in SEQ ID NO:38, CDR2 shown in SEQ ID NO:39, and CDR3 shown in SEQ ID NO:40, preferably, the antigen-binding region specifically binding to LILRB4 comprises the amino acid sequence shown in SEQ ID NO:

41.

43. The CAR of claim 41, wherein the other antigen is CD19, and the antigen-binding region specifically binding to CD19 comprises the heavy chain variable region (VH) shown in SEQ ID NO:42 and the light chain variable region (VL) shown in SEQ ID NO:43, preferably, the antigen-binding region specifically binding to CD19 comprises the amino acid sequence shown in SEQ ID NO:

44.

44. A chimeric antigen receptor (CAR) targeting BCMA, wherein the CAR comprises the amino acid sequence shown in SEQ ID NO:79 or 80.

45. An isolated therapeutic immune cell comprising the STAR of any one of claims 9-38 or the CAR of any one of claims 39-44.

46. ​​The therapeutic immune cell of claim 45, wherein the immune cell is a T cell or an NK cell, preferably a T cell.

47. The therapeutic immune cell of claim 45 or 46, wherein the therapeutic immune cell further comprises membrane-bound IL-15 protein (mbIL-15).

48. The therapeutic immune cell of claim 47, wherein mbIL-15 is a fusion protein formed by linking the extracellular domains of IL-15 and IL-15Ra (e.g., via a linker).

49. The therapeutic immune cells according to claim 48, wherein... i) The amino acid sequence of IL-15 is shown in SEQ ID NO:45; ii) The amino acid sequence of the extracellular domain of IL-15Ra is shown in SEQ ID NO:46; iii) The amino acid sequence of the linker connecting the extracellular domain of IL-15Ra to IL-15 is shown in SEQ ID NO:47; and / or iv) The amino acid sequence of mbIL-15 is shown in SEQ ID NO:

48.

50. An expression vector comprising the coding sequence of STAR of any one of claims 9-38 or CAR of any one of claims 39-44, preferably, the expression vector further comprising the coding sequence of a membrane-bound IL-15 protein (mbIL-15).

51. The expression vector according to claim 50, wherein mbIL-15 is a fusion protein formed by linking the extracellular domains of IL-15 and IL-15Ra (e.g., via a linker), preferably, wherein i) The amino acid sequence of IL-15 is shown in SEQ ID NO:45; ii) The amino acid sequence of the extracellular domain of IL-15Ra is shown in SEQ ID NO:46; iii) The amino acid sequence of the linker connecting the extracellular domain of IL-15Ra to IL-15 is shown in SEQ ID NO:47; and / or iv) The amino acid sequence of mbIL-15 is shown in SEQ ID NO:

48.

52. The expression vector according to claim 50 or 51, wherein the expression vector comprises a) The encoding nucleotide sequence of a fusion polypeptide comprising the α chain and the β chain of the STAR linked by a self-cleaving peptide; b) A nucleotide sequence encoding a fusion polypeptide comprising the α chain of the STAR, the β chain of the STAR, and the mbIL-15 linked by a self-cleaving peptide; or c) The encoding nucleotide sequence of the fusion polypeptide comprising the CAR and the mbIL-15 linked by a self-cleaving peptide.

53. The expression vector according to claim 52, 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:

37.

54. The expression vector according to claim 52 or 53, 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; ii) The fusion polypeptide comprises, from the N-terminus to the C-terminus, 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; or iii) The fusion polypeptide may include the CAR, a self-cleaving peptide such as Furin-P2A, and the mbIL-15 from the N-terminus to the C-terminus.

55. A method for preparing therapeutic immune cells, comprising: Step 1) Provide initial immune cells; Step 2) Introduce the expression vector of any one of claims 50-54 into the initiating immune cells; and Step 3) Harvest the immune cells obtained in Step 2).

56. The method of claim 55, wherein the initiating immune cell is a T cell or an NK cell, preferably a T cell.

57. Therapeutic immune cells that can be obtained or acquired by the expression vector of any one of claims 50-54 or the method of any one of claims 55-56.

58. A pharmaceutical composition comprising a single-domain antibody of any one of claims 1-4, a therapeutic immune cell of any one of claims 45-49 and 57, and / or an expression vector of any one of claims 6 and 50-54, and a pharmaceutically acceptable carrier, preferably for use in treating a disease in a subject.

59. Use of the single-domain antibody of any one of claims 1-4, the therapeutic immune cell of any one of claims 45-49 and 57, the expression vector of any one of claims 6 and 50-54, and / or the pharmaceutical composition of claim 58 in the preparation of a medicament for treating a disease in a subject.

60. A method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of a single-domain antibody of any one of claims 1-4, a therapeutic immune cell of any one of claims 45-49 and 57, an expression vector of any one of claims 6 and 50-54, and / or a pharmaceutical composition of claim 58.

61. The pharmaceutical composition of claim 58, the use of claim 59, or the method of claim 60, wherein the disease is a BCMA-related disease, such as a BCMA-related cancer, preferably, the cancer is myeloma, such as multiple myeloma (MM), particularly relapsed or refractory multiple myeloma (RRMM); or an autoimmune disease, such as systemic lupus erythematosus (SLE), myositis, scleroderma, Sjögren's syndrome, autoimmune hemolytic anemia, or rheumatoid arthritis.

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