Nanobodies targeting tacis and uses thereof

Abstract

The application provides a nanobody targeting human transmembrane activator and calcium modulator and cyclophilin interactor (TACI) and an application thereof, and the antibody is derived from a llama heavy chain antibody variable region. The anti-TACI single-domain antibody NB38 can specifically bind to human TACI with high affinity, can recognize a recombinant TACI protein, can also recognize a TACI with a natural conformation on a cell surface, and can partially block a BAFF / APRIL signal pathway. Based on the nanobody, the application constructs a fusion protein, a chimeric antigen receptor, an effector cell expressing the chimeric antigen receptor, and a double-specific cell linker and other genetically engineered forms. The nanobody can be further extended into a drug coupling form. The product of the application can be used for mediating directional recognition and killing of TACI positive cells, and can be used as a supplement and expansion of BCMA targeted therapy, and provides a new candidate technical scheme for targeted therapy of multiple myeloma and other TACI related diseases.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and tumor immunotherapy technology, specifically relating to a nanobody that targets and binds to TACI or its antigen-binding fragment, as well as nucleic acids, expression vectors, fusion proteins, chimeric antigen receptors, bispecific cell connectors and their applications encoding the nanobody or its antigen-binding fragment. The nanobody can also be further extended to a drug-conjugated form. Background Technology

[0002] Transmembrane activator and CAML Interactor (TACI), also known as tumor necrosis factor receptor superfamily member 13B (TNFRSF13B), is mainly expressed in B cell lineage cells and participates in humoral immune regulation. TACI can bind to B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL), playing a crucial role in B cell homeostasis, plasma cell differentiation, and survival.

[0003] In the treatment of multiple myeloma (MM), BCMA (B Cell Maturation Antigen) has become a mainstream target, but antigen escape, decreased antigen density, and drug resistance relapse remain recognized challenges. Studies have shown that some patients who relapse after BCMA-targeted therapy still retain TACI expression in their tumor cells, making TACI a highly promising alternative or complementary target.

[0004] In existing technologies, therapeutic molecules targeting TACI mainly focus on TACI-Fc fusion proteins or traditional monoclonal antibodies, as well as a small number of CAR-T cells based on traditional antibody fragments. However, these molecules suffer from problems such as large molecular weight, limited tissue penetration, and insufficient flexibility in engineering modification. Nanobodies (VHHs) have advantages such as small molecular weight, structural stability, and ease of engineering modification, but there is still a lack of nanobodies that can bind to TACI with high affinity and specificity and are suitable for development on various engineered therapeutic platforms, such as CAR-T, TCE, and further expandable drug-drug conjugates.

[0005] Therefore, developing a novel, multi-platform compatible, high-affinity anti-TACI nanobody is of significant clinical value in overcoming the limitations of existing BCMA-targeted therapies and providing new treatment strategies for TACI-related diseases such as multiple myeloma. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a nanobody capable of specifically binding to TACI and its antigen-binding fragment, and constructs various engineered applications based on this core. The anti-TACI nanobody NB38 obtained by this invention can bind to human TACI with high affinity and specificity. It can recognize not only recombinant TACI protein but also natively conformed TACI molecules on the surface of tumor cells, and can partially block the binding of BAFF / APRIL to TACI. Based on this nanobody, this invention further constructs various functional molecules such as fusion proteins, chimeric antigen receptors (CARs), immune effector cells expressing the CAR, and bispecific cell connectors (TCEs). The nanobody can also be further extended into drug-conjugated forms. The above products can be used to mediate the targeted recognition and killing of TACI-positive cells and can serve as candidate technologies for TACI-targeted therapy in multiple myeloma, especially as a supplement and expansion of BCMA-targeted therapy.

[0007] This invention is mainly achieved through the following technical solutions:

[0008] One aspect of the present invention provides a nanobody or antigen-binding fragment thereof targeting TACI, which satisfies one of the following:

[0009] (1) Contains the amino acid sequence shown in SEQ ID NO:1;

[0010] (2) Consists of the amino acid sequence shown in SEQ ID NO:1; and

[0011] (3) Contains an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:1, and contains CDR1 as shown in SEQ ID NO:18 (DYGMG), CDR2 as shown in SEQ ID NO:19 (GISWNAGSTYYAESVNG) and CDR3 as shown in SEQ ID NO:20 (VPPQGN).

[0012] One aspect of the present invention provides an isolated nucleic acid molecule encoding the aforementioned nanobody or its antigen-binding fragment.

[0013] One aspect of the present invention also provides an expression vector comprising the above-described nucleic acid molecule or expressing the above-described nanobody or its antigen-binding fragment.

[0014] One aspect of the present invention also provides a host cell comprising the above-described nucleic acid molecules, or expression vectors, or capable of expressing the above-described nanobodies or their antigen-binding fragments.

[0015] Another aspect of the present invention provides a fusion protein comprising the above-described nanobody or its antigen-binding fragment.

[0016] In some embodiments, the fusion protein further comprises one or more of the following: an Fc domain, an albumin-binding domain, a long-acting peptide, and a tag sequence linked to the nanobody or its antigen-binding fragment. Optionally, the Fc domain may be derived from human IgG1, IgG2, IgG4, or engineered variants thereof.

[0017] In another aspect, the present invention provides a chimeric antigen receptor comprising an antigen recognition domain, wherein the antigen recognition domain comprises the aforementioned nanobody or its antigen-binding fragment.

[0018] In some embodiments, the chimeric antigen receptor further includes a transmembrane region and an intracellular signal transduction domain.

[0019] In some embodiments, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, the following: a signal peptide; an antigen recognition domain; a hinge region and / or a spacer region; a transmembrane region; and an intracellular signal transduction domain. Optionally, the intracellular signal transduction domain comprises CD3ζ and further comprises one or more co-stimulatory domains selected from CD28, 4-1BB, OX40, ICOS, DAP10, and DAP12.

[0020] In some embodiments, the intracellular signal transduction domain contains the ITAM motif, which is a major driving part of TCR signaling and can activate T cells after CAR binds to target cells. Intracellular signal transduction domains known in the art can be used; in one embodiment of the present invention, it is the CD3ζ (Uniprot: P20963) intracellular signal transduction domain, the amino acid sequence of which is shown in SEQ ID NO:21.

[0021] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 21).

[0022] In some embodiments, the hinge region is used to provide sufficient flexibility and spatial distance to allow the antigen recognition domain to extend well out of the cell surface and bind to TACI. Hinge region amino acid sequences or flexible linkers known or common in the art can be used, such as the CD8α hinge region, the CD28 extracellular hinge region, (Gly-Ser) repeatable linker peptides, etc. One embodiment of the invention is a human CD28 (Uniprot: P10747) extracellular hinge region, the amino acid sequence of which is shown in SEQ ID NO:22.

[0023] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 22).

[0024] In some embodiments, the transmembrane region is used to anchor the receptor to the cell membrane and influence receptor polymerization and signal transduction. Amino acid sequences of transmembrane regions known or common in the art can be used, such as the CD8α transmembrane domain, the CD28 transmembrane domain, etc. One embodiment of the invention includes a human CD28 transmembrane domain, the amino acid sequence of which is shown in SEQ ID NO:23.

[0025] FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 23).

[0026] In some embodiments, the co-stimulatory domain provides a co-stimulatory signal to enhance the activation, proliferation, persistence, and cytotoxic function of immune effector cells. Co-stimulatory domains known in the art can be used, including but not limited to CD28, 4-1BB (CD137), OX40, CD27, ICOS (CD278), DAP10, DAP12, 2B4, and combinations thereof. One embodiment of the present invention is a human 4-1BB (Uniprot: Q07011) intracellular co-stimulatory domain, the amino acid sequence of which is shown in SEQ ID NO:24.

[0027] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 24).

[0028] In some embodiments, the chimeric antigen receptor further includes a second antigen recognition domain that targets and binds to BCMA.

[0029] In some embodiments, the chimeric antigen receptor is a tandem dual-target CAR, a bicistronic CAR, a dual-vector co-transduction CAR, or a CAR system that is co-administered with BCMA-CAR.

[0030] In another aspect, the present invention provides an immune effector cell that expresses the above-described chimeric antigen receptor.

[0031] In some embodiments, the immune effector cells are selected from one or more of the following: γδT cells, αβT cells, peripheral blood T cells, NK cells, NKT cells, cytotoxic T lymphocytes, iPSC-derived immune cells, macrophages, dendritic cells, and combinations thereof; preferably γδT cells.

[0032] In another aspect, the present invention provides a bispecific or multispecific cell connector, comprising:

[0033] (1) Tumor recognition end, which contains the above-mentioned nanobody or its antigen-binding fragment;

[0034] (2) Immune cell binding end, which can bind to molecules on the surface of immune effector cells.

[0035] In some embodiments, the immune cell binding terminus can bind γδTCR, CD3, CD16, NKp30, NKp46, NKG2D, or combinations thereof.

[0036] In some embodiments, the cell connector further includes a second tumor recognition end that targets and binds to BCMA.

[0037] In another aspect, the present invention provides a pharmaceutical composition comprising one or more of the above-described nanobody or its antigen-binding fragment, fusion protein, chimeric antigen receptor, immune effector cell, and cell connector, as well as a pharmaceutically acceptable carrier or excipient.

[0038] The use of the aforementioned nanobodies or their antigen-binding fragments, nucleic acid molecules, expression vectors, host cells, fusion proteins, chimeric antigen receptors, immune effector cells, cell connectors, or pharmaceutical compositions in the preparation of medicaments for treating TACI-positive diseases is also within the scope of this invention.

[0039] In some embodiments, the TACI-positive disease is multiple myeloma, plasma cell tumor, B-cell lymphoma, autoimmune disease, or a combination thereof.

[0040] In some embodiments, the multiple myeloma is a multiple myeloma with heterogeneous BCMA expression, decreased BCMA expression, or failure of BCMA-related treatment.

[0041] In some embodiments, the drug is used in combination with or sequentially with BCMA-targeting drugs, BCMA-CAR, BCMA-TCE, proteasome inhibitors, immunomodulators, monoclonal antibodies, chemotherapeutic drugs, radiotherapy, or combinations thereof.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) A novel high-affinity TACI nanobody is provided. The NB38 nanobody obtained by this invention can achieve nanomolar affinity (KD=2.77×10⁻⁶). -9 M) specifically binds to human TACI and can effectively recognize the native conformation of TACI on the surface of tumor cells. At the same time, it can partially block the interaction between BAFF / APRIL and TACI, providing a high-quality core recognition molecule for TACI-targeted therapy.

[0044] (2) Excellent engineering adaptability and multi-platform applicability. The nanobody of the present invention has a simple structure and small molecular weight, making it easy to engineer. Experiments have shown that it can be successfully constructed into various functional molecules such as Fc fusion proteins, chimeric antigen receptors (CARs), and bispecific cell connectors (TCEs), demonstrating good platform expansion capabilities. In particular, the NB38-TACI CAR constructed based on NB38 achieved a transduction positivity rate of 68.7% in activated and expanded αβT cells, and the BCMA / TACI dual-target CAR also maintained a transduction positivity rate of 61.6%, indicating that the CAR construct based on the VHH recognition domain has good lentiviral transduction adaptability and expression feasibility.

[0045] (3) It can serve as a supplement and extension to BCMA-targeted therapy, providing a new candidate intervention pathway to address tumor antigen heterogeneity. This invention focuses on TACI as a supplementary or alternative target for multiple myeloma, and experimentally verified the clear in vitro and in vivo antitumor activity of CAR-γδT cells constructed based on NB38 in TACI-positive multiple myeloma.

[0046] (4) Adaptation to novel immune effector cells. This invention is not only applicable to traditional αβT cells, but also successfully constructed NB38-based γδT CAR-T and TCEs that recruit γδT cells. By utilizing the unique innate immune characteristics and tumor homing ability of γδT cells, effective killing of TACI-positive tumor cells was achieved, expanding the application scope of immunocellular therapy.

[0047] (5) Forming a dual-target strategy that complements BCMA. Dual-target CARs have shown a stronger trend in tumor control in double-positive tumor models, providing a new technical solution for addressing tumor antigen heterogeneity and reducing the risk of single-target escape.

[0048] (6) It has the potential to be used in both tumor therapy and autoimmune diseases. Since TACI is located at a critical position on the BAFF / APRIL axis, the nanobody and its engineered form of the present invention can not only be used for tumor targeted therapy, but also provide new tool molecules for the development of blocking molecules for autoimmune diseases. Attached Figure Description

[0049] Figure 1 The image shows the results of detecting the TACI-specific antibody titer in immunized alpaca plasma.

[0050] Figure 2 The image shows the detection results for screening positive monoclonal strains of TACI phage.

[0051] Figure 3 The image shows the SDS-PAGE Coomassie Brilliant Blue staining results of 11 recombinant TACI-VHH-IgG4 Fc fusion proteins under reducing and non-reducing conditions.

[0052] Figure 4 The figure shows the ELISA results of the binding activity of 11 recombinant TACI-VHH-IgG4 Fc fusion proteins to TACI protein.

[0053] Figure 5 The image shows the BLI assay results for the binding affinity of NB38-Fc to human TACI protein.

[0054] Figure 6 The image shows the detection results of the blocking effect of NB38-Fc on the binding of TACI to BAFF / APRIL.

[0055] Figure 7 This is a schematic diagram of the molecular docking between NB38 and the extracellular domain (1-166aa) of human TACI.

[0056] Figure 8 Flow cytometry results for NB38-Fc to identify TACI on the surface of multiple myeloma cells.

[0057] Figure 9 Schematic diagram of the structure designed for different chimeric antigen receptor CARs.

[0058] Figure 10 The results of flow cytometry analysis of EGFP in untransduced T cells, NB38-TACI CAR-T, BCMA CAR-T, and BCMA / TACI dual-target CAR-T were 0.25%, 68.7%, 74.4%, and 61.6%, respectively.

[0059] Figure 11 Figure 1 shows the results of in vitro cytotoxicity assays of different CAR-γδT inhibitors on multiple myeloma cells.

[0060] Figure 12 The figure shows the detection results of cytokine release levels after co-culturing different CAR-γδT cells with tumor cells.

[0061] Figure 13 This is a schematic diagram of the timeline of a xenograft mouse model.

[0062] Figure 14 These are representative images of in vivo bioluminescence imaging at different time points in each group of xenograft mouse models.

[0063] Figure 15 The results are a quantitative analysis of the average whole-body luminescence intensity of each xenograft mouse model.

[0064] Figure 16 The diagram shows the structures of γδTCE molecules with three different structural arrangements.

[0065] Figure 17 The results of SDS-PAGE analysis of three γδTCE fusion proteins with different structural arrangements are shown.

[0066] Figure 18 The image shows the detection results of in vitro expansion of γδT cells using γδTCE molecules with different configurations.

[0067] Figure 19 The figure shows the detection results of the bridging effect of γδTCE molecules in the co-culture system. Detailed Implementation

[0068] To better understand the technical solution of this invention, the following detailed description is provided through specific embodiments. These embodiments are only for explaining this invention and should not be construed as limiting the invention in any way. Those skilled in the art can make adjustments or equivalent substitutions to the implementation details without departing from the spirit of this invention, all of which fall within the protection scope of this invention.

[0069] In this invention, peripheral blood lymphocyte RNA was obtained by immunizing camel-derived animals, and the VHH gene fragment was obtained by reverse transcription, and a phage display library was constructed. Using recombinant human TACI protein as an antigen, multiple rounds of specific screening yielded several heavy-chain single-domain antibodies with high affinity for TACI. Sequencing analysis showed that each antibody's VHH fragment possessed typical framework region (FR1-FR4) and complementarity-determining region (CDR1-CDR3) structures, with different amino acid sequences and independent recognition epitopes. The selected candidate nanobodies exhibited significant activity in in vitro binding experiments, cell signal blocking, and immune cell co-culture models, demonstrating their feasibility for targeted therapy.

[0070] The following embodiments are used to further illustrate the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the present invention. Equivalent substitutions and improvements made by those skilled in the art without departing from the spirit of the present invention should all be included within the scope of the present invention.

[0071] Example 1: Detection of plasma-specific antibody titer in alpacas immunized with human TACI protein

[0072] This embodiment uses recombinant human TACI protein to immunize alpacas and detects whether specific antibodies against TACI protein are produced in the plasma of immunized alpacas. The specific steps include the following.

[0073] 1.1) A 4-year-old male alpaca (purchased from an Inner Mongolia farm) was immunized with recombinant TACI-Fc protein (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number 29965-H02H), which is a fusion expression of the extracellular fragment of human TACI and the IgG1-Fc fragment. The alpaca underwent a primary immunization and three booster immunizations. For the primary immunization, 0.5 mg of recombinant human TACI protein was mixed with complete Freund's adjuvant in a 1:1 ratio and thoroughly emulsified, and then injected subcutaneously. Subsequently, 0.25 mg of recombinant human TACI protein was mixed with incomplete Freund's adjuvant in a 1:1 ratio for three booster immunizations. A total of four immunizations were administered, with each immunization occurring two weeks apart.

[0074] 1.2) Three days after the last immunization, 100 ml of whole blood was collected from alpacas via intravenous infusion. The whole blood was centrifuged, and the supernatant plasma was collected to detect the production of recombinant human TACI protein-specific antibodies (hereinafter also referred to as TACI protein-specific antibodies). Specifically, the supernatant plasma (initial dilution of 1:500) was serially diluted to obtain 12 concentrations of test samples. The titer of TACI protein-specific antibodies in these samples was detected using the ELISA method (detailed below). The lower layer of whole blood cells was diluted with 1640 medium, and PBMCs (peripheral blood mononuclear cells) were isolated to establish the VHH gene library in subsequent examples.

[0075] The titer detection of TACI protein-specific antibodies in alpaca plasma (based on ELISA principles) mainly includes the following procedures.

[0076] (1) The recombinant human His-tagged TACI extracellular fragment (1-166aa) protein (purchased from Beijing ACRO Biotechnology Co., Ltd., catalog number: TAI-H52H3) was diluted to 1 μg / mL with pH 9.6 0.1 mol / L NaHCO3 coating buffer and added to 96 microplates, 100 μL per well, and coated overnight at 4℃.

[0077] (2) Wash twice with 0.1% TBST, add 300 μL of TBST blocking solution containing 3% BSA, and incubate at 37°C for 2 h.

[0078] (3) Add the above serially diluted alpaca plasma, set up 2 sub-wells for each dilution, and incubate at 37°C for 2 hours.

[0079] (4) Washing and adding enzyme-labeled secondary antibody: Dilute HRP-labeled anti-allama antibody Goat Anti-Llama IgG H&L (HRP) (Abcam, catalog number: ab112786) 1:8000, 100 μL / well, and incubate at 37℃ for 1 h.

[0080] (5) Add 100 μL / well TMB colorimetric solution, incubate at 37℃ for 5 min, add 100 μL / well 2 mol / L H2SO4 stop solution to terminate the colorimetric reaction, and measure the absorbance value using an enzyme-linked immunosorbent assay (ELISA) reader. The absorption wavelength is 450 nm and the correction wavelength is 630 nm.

[0081] The results are as follows Figure 1 As shown, alpaca plasma before immunization served as a negative control. It was observed that high-titer antibodies against TACI protein were produced in the alpaca plasma after immunization, and this titer increased with increasing plasma dilution (e.g., dilution greater than 10). 5 The reaction value decreased, showing a dose-dependent effect. This result suggests that alpacas immunized four times did indeed produce high-titer specific antibodies against the TACI protein, which can be used for VHH gene library construction and nanobody screening. The detection antigen and the immune antigen use different tag formats, which helps reduce interference from non-specific recognition of Fc fragments or tags.

[0082] Example 2: Screening for phage-displayed nanobodies that specifically bind to human TACI protein

[0083] This embodiment first performs alpaca immunization and VHH gene library construction, and then uses phage display technology to screen nanobodies that can specifically bind to TACI protein from PBMCs in alpaca whole blood cells immunized with TACI protein. The specific operations include the following steps.

[0084] 2.1) Construction of the alpaca immune VHH gene library

[0085] 2.1.1) Collect the PBMCs obtained from the lower layer of whole blood cells in Example 1 (1.1) using Ficoll-Paque PLUS lymphocyte separation medium (GE, catalog number 17-1440-03). Total RNA was extracted using RNA extraction reagent (RNAiso Plus), and synthetic cDNA was obtained using the PrimeScrip II reverse transcription kit (TAKARA, catalog number 6210A).

[0086] 2.1.2) Using the cDNA obtained above as a template, nested PCR was used to amplify the VHH gene fragment of the heavy chain antibody variable region. Subsequently, the obtained PCR product was subjected to agarose gel electrophoresis, and bands <500 bp were collected and purified. After PCR amplification and purification, the expression sequence of the heavy chain variable region (VHH) fragment of the antibody naturally lacking the light chain in alpaca peripheral blood was obtained.

[0087] 2.1.3) The purified nested PCR product (400 bp amplification band) and the phage pComb3x vector were digested with restriction endonuclease SfiⅠ. The digested products were then ligated at a molar ratio of 3:1 in the presence of T4 ligase at 16°C and electroporated into Escherichia coli XL1-blue competent cells.

[0088] The electroporated competent cells were incubated in 2×YT medium at 37°C with shaking for 1 hour. The transformation solution was then used to incubate the cells at 10... 3 10 6 10 9 The volume of the *E. coli* clone library was determined by dilution and plated with Amp-resistant, IPTG- and X-gal-treated plates. The remaining transformation solution was plated on a large scale and incubated overnight at 37 °C. The next day, the colonies were scraped and combined to prepare the VHH library stock solution. The positive cloning efficiency (i.e., VHH fragment insertion rate) of the VHH fragment was identified. Based on blue-white screening, the positive cloning rate was calculated using the following formula: Library size (CFU) = Colony count × Dilution factor × Positive cloning rate × (Total transformation volume / Plate volume). The calculated library size of the alpaca phage VHH display gene library was approximately 2.79 × 10⁻⁶. 9 CFU, insertion accuracy rate 98%.

[0089] 2.2) Amplification of VHH gene library phages

[0090] XL1-blue cells transformed with the VHH gene library phage (obtained in step 2.1) were seeded in 10 ml of SOC-Amp medium and cultured at 37°C and 200 rpm for 3 h to induce a pre-log phase with an abundance of 8 × 10⁻⁶ cells / year. 8Add M13K07 helper phage at a multiplicity of infection (MOI) of 20–30 cells / mL. Incubate at 37°C for 30 min. Centrifuge XL1-blue cells at 2800×g for 10 min at room temperature. Resuspend the XL1-blue cell pellet in 50 mL LB-Amp medium, add 100 μg / mL Amp antibiotic and 50 μg / mL kana antibiotic, and incubate at 37°C with shaking at 200 rpm for 8 h. Transfer the overnight culture to a 50 mL centrifuge tube and centrifuge at 3200×g for 15 min at 4°C. Transfer the supernatant to another centrifuge tube and centrifuge again at 3200×g for 15 min at 4°C. Take 80% of the supernatant from the top of the centrifuge tube, transfer it to a new centrifuge tube, add 1 / 5 volume of PEG6000 / 2.5 mol / L NaCl, and incubate at 4°C overnight to precipitate. The following day, the centrifuge tube was centrifuged at 3200×g for 15 min at 4℃, the supernatant was discarded, and the remaining liquid was removed after centrifugation for 30 s. The phage precipitate was collected on the side wall of the tube. The precipitate was resuspended in 1 mL PBS and transferred to a microcentrifuge tube, centrifuged at 20,000×g for 1 min at 4℃. The supernatant was transferred to a new centrifuge tube, and the phage was reprecipitated again with 1 / 5 volume of PEG6000 / 2.5 mol / L NaCl, and incubated on ice for 60 min. The tube was then centrifuged at 20,000×g for 15 min at 4℃, the supernatant was discarded, and the remaining liquid was removed after centrifugation for 30 s. The precipitate was resuspended in 500 μL PBS and centrifuged for 1 min. The supernatant was transferred to a new EP tube, which contained the amplified VHH phage display library. The titer was determined to be 9×10⁻⁶. 9 pfu / mL, used for subsequent specific VHH screening.

[0091] 2.3) Screening of TACI protein-specific VHH phages

[0092] A solid-phase screening method was used to select TACI protein specifically binding to VHH. TACI protein was diluted to a final concentration of 100 μg / mL with coating buffer and added to 96-well plates, 100 μL per well. The 96-well plates were placed in a humidified chamber and incubated overnight at 4°C. The coating buffer in the 96-well plates was discarded, and the plates were washed twice rapidly with 0.1% PBST (PBS + 0.1% Tween 20). 300 μL of 1% BSA blocking buffer was added to each well, and the plates were blocked at 4°C for 2 h. 10 μL of the phage library stock solution was mixed with 100 μL of PBST and added to each well, 100 μL / well, and incubated at room temperature with gentle shaking for 30 min. The plates were washed at least 5 times with 0.1% PBST to remove non-specifically bound phages. Specifically bound phages were eluted with Gly-HCl (pH 2.2), and the resulting phages were infected with logarithmic-phase XL1-blue and amplified for the next round of screening. After four rounds of "adsorption-elution-amplification," a phage library enriched with antigen-positive antibodies was obtained through screening. The phages specifically enriched in the final round were directly used for plating and clone picking.

[0093] 2.4) TACI protein specifically binds to antibodies expressed by monoclonal phages (ELISA principle)

[0094] Dissolve TACI protein in coating buffer at a concentration of 1 μg / mL, add 100 μL to each well of a 96-well ELISA microplate, and coat overnight at 4°C. Wash twice with 0.1% TBST. Add 300 μL of PBST blocking buffer containing 3% BSA. Use uncoated TACI protein plates as controls. Incubate at 37°C for 2 h for all blocking conditions. Add 100 μL of monoclonal phage culture supernatant to each well coated with the target protein, and add 100 μL to each uncoated well as a control. Incubate at 37°C for 2 h. Wash 6 times with PBST. Add 100 μL of HRP-labeled anti-M13 monoclonal antibody (catalog number: 11973-MM05T-H) diluted 1:8000 with PBST to each well, and incubate at 37°C for 1 h. Wash 6 times with PBST. Add substrate chromogenic buffer (TMB) and develop for 20 min (100 μL / well). Stop the reaction with 2 mol / L H₂SO₄ (100 μL / well). Measure the absorbance using a microplate reader at 450 nm (corrected wavelength: 630 nm). Clones with absorbance values ​​at least 5 times greater than the negative control and an OD value greater than 1 are considered positive. Select positive clones with high OD values ​​and send them to a sequencing company for sequencing to obtain their nucleotide sequences. This example involved four rounds of selection, yielding 280 positive clones with an OD value greater than 1 from 400 single clones (ELISA results are shown below). Figure 2As shown in the figure, the OD values ​​were sorted, and the top 200 positive monoclonal strains were selected for PCR amplification. The specific VHH gene sequence of each positive clone was obtained by sequencing, and the corresponding VHH amino acid sequence was obtained from it.

[0095] This study obtained 14 unique VHH amino acid sequences from 200 selected positive monoclonal strains, and defined and compared their complementarity-determining regions (CDRs). The CDRs were delineated according to both the Kabat and Chothia classic antibody numbering systems. Specifically, CDR1 and CDR2 were identified according to the boundaries defined by their respective numbering standards; CDR3, based on the variable region sequence characteristics of nanobody, was the hypervariable region sequence from the conserved cysteine ​​residue at the FR3 terminal to the initiation of FR4. Based on these rules, Kabat CDR1, CDR2, and CDR3, as well as Chothia CDR1, CDR2, and CDR3, were extracted from each sequence for subsequent sequence comparison and structural feature analysis. In the 14 nanobody sequences in this study, the CDR3 delineation results under both the Kabat and Chothia standards were consistent; the main difference lay in the boundary delineation of CDR1 and CDR2. The 14 nanobody sequences were classified into CDRs according to the Kabat and Chothia criteria, and the results are shown in Table 1.

[0096] Table 1: Amino acid sequence information of 14 TACI nanobodies

[0097]

[0098] Example 3: Expression, purification, and detection of binding properties of nanobody-Fc fusion protein

[0099] The nanobody sequences obtained in Example 2 (see Table 1) were ligated with the human IgG4 Fc sequence to construct the expression vector pcDNA3.4. The fusion protein, from N-terminus to C-terminus, includes a signal peptide sequence (SEQ ID NO:15), a nanobody sequence (see Table 1), and a human IgG4 Fc sequence (SEQ ID NO:16).

[0100] Specifically, the following steps are included:

[0101] (1) Preparation of VHH fragment: VHH fragment was obtained by PCR. The VHH fragment was added to membrane binding buffer, mixed thoroughly, and then added to the purification column. After centrifugation at 13000 rpm for 1 min, the liquid was discarded. 700 μL of washing buffer was added, and after centrifugation at 13000 rpm for 1 min, the liquid was discarded. After centrifugation for 5 min, the column was placed at room temperature for 5 min, and then Nuclease-Free Water was added to elute, thus obtaining the purified VHH fragment.

[0102] (2) Cloning: 4 μL of purified fragment, 1 μL of linearized expression vector, and 5 μL of DNA ligase were added and thoroughly mixed. After reacting at 50℃ for 20 min, the mixture was added to 100 μL of DH5α competent cells, incubated on ice for 30 min, followed by heat shock for 90 s. The cells were then quickly placed on ice for 3 min and plated, and cultured overnight at 37℃. The obtained single clones were amplified and sequenced to obtain the expression nanobody and Fc fusion protein vector. The plasmid was extracted and transfected into HEK293T cells for transient expression. HEK293T cells were passaged in serum-free HEK293T medium, and serum-free medium was added on days 1, 3, and 5 after transfection. Shake flask culture conditions: 5% CO2, temperature 37℃, shaker speed 175 rpm.

[0103] (3) Collect the cell culture medium aseptically, centrifuge at 1000 g for 20 min using a benchtop centrifuge, collect the supernatant, filter it through a 0.22 μm filter membrane, and use ÄKTA. TM The Advantage 25 purification system was used to purify the fusion protein of IgG4-Fc using the Protein A Trap Column. Specific steps:

[0104] 1) Water balance: Rinse 2CV with ultrapure water to replace the 25% ethanol preservation solution;

[0105] 2) Equilibration column: AC binding equilibrates 3CV until the UV baseline is stable;

[0106] 3) Sample loading: Adjust the appropriate flow rate for sample loading; rinsing: Rinse with rinsing solution for 5-10 CV until the UV baseline is stable, at the same flow rate as the sample loading flow rate;

[0107] 4) Elution: Elutin was used for elution, and samples were collected based on the UV peak;

[0108] 5) Neutralization: Add Tris and neutralize the eluted antibody at pH 8.0;

[0109] 6) Balance: AC binding balances 3CV to neutral;

[0110] 7) CIP cleaning: CIP cleaning with 5CV or higher;

[0111] 8) Rinse with alkali: Rinse with AC binding until the pH at the outlet is neutral, then rinse with ultrapure water for 3CV; 9) Storage: Equilibrate with 25% ethanol for 2CV, and store the column.

[0112] (4) The supernatant was purified by Protein A affinity chromatography and dialyzed into PBS buffer to obtain purified nanobody-Fc fusion protein.

[0113] The concentration of the fusion protein was quantitatively detected using a micro-spectrophotometer. The sample was spotted with the corresponding buffer solution. A baseline stability was indicated when the absorbance value was within ±0.015 at 280 nm. The purified sample was then spotted sequentially, and the absorbance values ​​were recorded. The concentration of the fusion protein (mg / mL) was obtained by dividing the detection data by the extinction coefficient of IgG (1.414).

[0114] The purity of the purified fusion protein was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This method separates proteins in the electrophoresis gel based on their molecular weight, thus verifying the purity of the target antibody. The specific steps for Coomassie brilliant blue staining are as follows: Add 5 μg of the purified antibody to 5 μL of 4×R or 4×N loading buffer, heat in a 100°C water bath, and then centrifuge at 10,000 rpm. Add 1×SDS electrode buffer to the electrophoresis tank, and use a micropipette to add the prepared sample solution and protein molecular weight standards to the wells. Connect the power supply and electrophores at a constant voltage of 100 V until the bromophenol blue dye enters the separating gel from the stacking gel. Then adjust the current to 120 V and continue electrophoresis until the bottom of the gel plate is reached. Turn off the power. Immerse the gel in the staining solution and stain at room temperature on a gently agitated platform. Replace the decolorizing solution to cover the gel, place it on a gently shaking platform at room temperature for decolorization, and repeat the decolorization process until a blue band and a clean background are obtained.

[0115] The binding specificity of these fusion proteins to TACI was detected using an ELISA method, specifically including the following steps: TACI protein (catalog number: TAI-H52H3) was diluted to a concentration of 0.1 μg / mL with coating buffer. 1 μg / mL was added to each well of a 96-well microplate at 100 μL / well, and the plate was incubated overnight at 4°C. The plate was washed twice with 0.1% TBST, and 300 μL of TBST blocking buffer containing 3% BSA was added. The plate was then blocked and incubated at 37°C for 2 h. The aforementioned numbered fusion protein was diluted to a concentration of 1 μg / mL, and 100 μL was added to each well, with two auxiliary wells. The plate was incubated at 37°C for 2 h. HRP-labeled anti-human IgG antibody was diluted 1:8000, and 100 μL was added to each well. The plate was incubated at 37°C for 1 h. Add 200 μL / well TMB chromogenic solution and incubate at 37℃ for 5 min. Stop the chromogenic reaction by adding 50 μL / well 2 mol / L H2SO4 stop solution. Measure the absorbance using a microplate reader. The absorption wavelength is 450 nm, and the calibration wavelength is 630 nm.

[0116] In Example 2, a total of 14 specific VHH sequences were obtained. The results in Example 3 showed that 11 of these 14 specific VHH sequences (numbered NB38, N848, N851, N864, N206, N212, N256, N269, N253, N265, and N341) were successfully constructed and expressed as a fusion protein of nanobodies and IgG4Fc, named TACI-VHH-IgG4Fc. Figure 3 The Coomassie Brilliant Blue staining results shown are as follows: A section shows the results for the fusion proteins N848, N851, and N864; B section shows the results for the fusion proteins N206, N212, N269, N253, and N341; and C and D sections show the results for the fusion proteins N256, N265, and NB38, respectively. It is evident that all 11 nanobodies exhibited clear target bands under both reducing and non-reducing conditions, with minimal impurities, indicating good protein expression and purification efficiency. In non-reducing SDS-PAGE… Figure 3 Under NR conditions, the TACI-VHH-IgG4Fc fusion protein exhibits a band of approximately 74-90 kDa, while under reduced SDS-PAGE (Non-NR) conditions, the molecular weight of the TACI-VHH-IgG4Fc fusion protein is significantly different. Figure 3 Under suitable conditions (R), the molecular weight is approximately 39-45 kDa, consistent with the molecular weight characteristics of Fc fusion protein dimers and monomers. Overall, all 11 TACI-VHH-IgG4Fc antibodies achieved stable expression and good purification, meeting the needs of subsequent functional testing and activity evaluation.

[0117] ELISA test results as follows Figure 4As shown, all 11 recombinant TACI-VHH-IgG4Fc antibodies exhibited varying degrees of TACI protein binding activity. The OD450 of the negative control (NC) well was close to zero, indicating a low background in the detection system. When the TACI protein coating concentration was 1 μg / mL, the OD450 values ​​of N269, N341, N253, N212, N206, N848, N851, N864, and NB38 were all high, indicating that these antibodies had a strong binding ability to TACI protein; among them, the binding signals of N848, N851, N864, N341, and NB38 were particularly strong. In contrast, the binding signals of N265 and N256 were relatively weaker, but still higher than those of the negative control, indicating that they still possessed some TACI binding activity.

[0118] When the concentration of the coated TACI protein decreased to 0.1 μg / mL, the binding signals of most antibodies decreased significantly, indicating that their detection signals were antigen concentration-dependent. However, NB38 maintained a high OD450 value, showing the strongest binding reaction. N341 and N851 also retained relatively detectable binding signals, while the signals of the remaining antibodies were generally weak. ELISA results showed that all 11 TACI-VHH-IgG4Fc antibodies obtained in this study could bind to TACI protein, but there were significant differences in binding strength among the different antibodies, with NB38 exhibiting significantly the best binding ability.

[0119] Example 4: Binding kinetics and blocking activity assay of exemplary TACI nanobody to human TACI protein

[0120] This embodiment aims to verify the binding kinetics between the nanobody and Fc fusion protein (taking the NB38 antibody prepared in Example 3 as an example, hereinafter referred to as NB38-Fc) and human TACI protein (catalog number: TAI-H52H3 as an example), and further evaluate its effect on the binding of TACI to the natural ligand BAFF / APRIL.

[0121] Specifically, detection was performed using the Fortebio Octet system based on bio-layer interferometry. The procedure involved immobilizing a His-tagged human recombinant TACI protein (5 μg / mL) with a pre-loaded nickel affinity matrix using an HIS sensor. After equilibration in binding buffer (PBS solution containing 0.02% Tween-20 and 0.1% BSA), the sensor was sequentially immersed in different concentrations of NB38-Fc antibody solutions (0, 3.125, 6.25, 12.5, 25, 50 nM) to record the binding process, followed by recording the dissociation process in antibody-free buffer. The resulting signal curves were fitted using Octet data analysis software according to a 1:1 binding model, and the results are shown below. Figure 5 As shown, the calculated binding rate constant (kon) is 1.49 × 10⁻⁶. 5 M -1 ·s -1 The dissociation rate constant (koff) is 4.12 × 10⁻⁶. -4 s -1 The equilibrium dissociation constant (KD) is approximately 2.77 × 10⁻⁶. -9 The results showed a clear and concentration-dependent binding signal between NB38-Fc and human TACI protein. The binding response value gradually increased with increasing antibody concentration; the dissociation signal decreased slowly, indicating that NB38-Fc exhibits a fast binding, slow dissociation, and high stability kinetic profile with TACI protein. These results suggest that NB38-Fc has a high affinity for human TACI protein at the nanomolar level. Further analysis using BAFF-R and BCMA proteins as controls yielded no significant binding signal, indicating that NB38-Fc has good specificity for the TACI target.

[0122] Further sequential binding experiments were used to evaluate the effect of NB38-Fc on the interaction between TACI and BAFF / APRIL. The sensor immobilized with TACI protein was first bound to NB38-Fc until the plateau phase, and then transferred to solutions containing BAFF-Fc (catalog number: 10056-H01H), APRIL-Fc (catalog number: 10610-h01h2), and a BAFF / APRIL mixed protein solution (equal proportions of BAFF-Fc and APRIL-Fc). Subsequent binding signals were recorded, with direct binding of the corresponding ligand to TACI serving as a control. Results are as follows: Figure 6As shown, after NB38-Fc pre-binding, BAFF-Fc, APRIL-Fc, and the BAFF / APRIL mixed protein can still bind to TACI, but their binding response is lower than that of the control group. This indicates that NB38-Fc has a partial blocking effect on the binding of BAFF / APRIL to TACI, rather than a completely competitive inhibition. This result suggests that the TACI epitope recognized by NB38-Fc may partially overlap with the binding region of BAFF / APRIL, or be located in its adjacent region and produce a certain steric hindrance effect.

[0123] Therefore, the nanobody NB38 described in this embodiment can bind to human TACI protein with high affinity and inhibit the binding of BAFF / APRIL to TACI to a certain extent. Furthermore, to analyze the potential binding mode of NB38 and TACI at the structural level, molecular docking was used to simulate the structure of NB38 and the extracellular domain (1-166aa) of human TACI. The results are as follows... Figure 7 As shown, the three complementarity-determining regions (CDR1, CDR2, and CDR3) of NB38 are all located near the binding interface with the extracellular domain of TACI, with CDR3 located in the major contact region. The two cysteine-rich domains (CRDs) of the TACI extracellular domain are located in its extracellular recognition region. These results suggest that NB38 may form a stable binding with the TACI extracellular domain through its CDR regions, supporting its high-affinity binding characteristics observed in in vitro experiments. Further analysis of the binding blockade experiment suggests that the epitope recognized by NB38 may be located near or partially overlap with the BAFF / APRIL binding region, thus creating a certain steric hindrance effect.

[0124] Example 5: Detection of TACI expression on the surface of an exemplary TACI nanobody recognizing human multiple myeloma cell line

[0125] This example aims to verify the recognition ability of the nanobody-Fc fusion protein (using NB38-Fc prepared in Example 3 as an example) on TACI on the surface of human multiple myeloma cell lines. The RPMI-8226, MM1S, and U266 cells used were all stably expressing Luc luciferase and RFP. All flow cytometry experiments were performed at 4°C. Specifically, RPMI-8226, MM1S, and U266 cells were resuscitated and cultured, and after collection, the cells were washed and resuspended in FACS buffer (PBS + 1% BSA) to adjust the cell concentration to 1×10⁻⁶. 6 / mL. First, add 5 μg / mL human Fc blocking reagent and incubate for 10 min to reduce Fc-mediated non-specific binding; then add recombinant NB38-Fc antibody to a final concentration of 10 μg / mL and incubate for 30 min. After washing, add anti-human IgG-Alexa Fluor 647 secondary antibody (1:400, Thermo Fisher Scientific, A21445) and incubate for 30 min, then wash again and perform flow cytometry detection. Simultaneously, to analyze the expression of BCMA on the surface of different multiple myeloma cell lines, flow cytometry was used to detect BCMA (APC-antiBCMA, clone number 19F2, catalog number 357506) in RPMI-8226, MM1S, and U266 cells. The fluorescence signal of BCMA on the cell surface was recorded in the APC channel to compare the expression characteristics of TACI and BCMA on the surface of each cell line.

[0126] RPMI-8226, MM1S, and U266 cells all stably expressed Luc / RFP, while significant fluorescence signals were detected in the PE channel of the blank control group. Therefore, this experiment mainly relied on changes in APC channel signal to determine the binding of NB38-Fc to TACI on the cell surface. Results are as follows: Figure 8 As shown, under blank control conditions, no significant positive shift was observed in the APC channels, indicating a low background in the experimental system and limited non-specific binding of the secondary antibody. After NB38-Fc staining, RPMI-8226 and MM1S cells showed a significant rightward shift in the APC channels, with a significant increase in the positive cell population, indicating that NB38-Fc can effectively bind TACI molecules on the surface of these two cell lines, suggesting that RPMI-8226 and MM1S are TACI-highly expressing cell lines. In contrast, although a certain degree of enhanced APC positive signal was observed in U266 cells after NB38-Fc staining, its overall shift amplitude and positive intensity were weaker than those of RPMI-8226 and MM1S, indicating that the TACI expression level on the surface of U266 cells was relatively low. Further analysis of surface BCMA molecule expression showed that RPMI-8226, MM1S, and U266 cell lines all exhibited significant BCMA positive signals, suggesting that they are all BCMA-highly expressing cell lines.

[0127] This experiment showed that RPMI-8226, MM1S, and U266 are all BCMA-high expression cell lines. RPMI-8226 and MM1S also showed high TACI expression, while U266 showed low TACI expression. These results indicate that NB38-Fc can not only bind to recombinant TACI protein but also effectively recognize native TACI molecules on the surface of multiple myeloma cells and distinguish expression differences between different cell lines. This result provides experimental evidence for subsequent research on NB38-based cell-targeted recognition, in vivo tracking, and targeted therapy.

[0128] Example 6: Construction and in vitro functional validation of an exemplary TACI-targeting chimeric antigen receptor

[0129] This embodiment aims to construct a chimeric antigen receptor based on the anti-TACI nanobody NB38 of the present invention, and to evaluate the in vitro antitumor activity of effector immune cells expressing the chimeric antigen receptor.

[0130] CAR Construction: A nucleic acid sequence encoding a CAR backbone polypeptide comprising, from N-terminus to C-terminus, the CD28 extracellular hinge region, the CD28 transmembrane region, the CD137 / 4-1BB co-stimulatory region, and the CD3ζ cytoplasmic region was obtained by chemical synthesis. This nucleic acid sequence was cloned downstream of a pre-modified lentiviral vector and operatively ligated to a constitutive human extension factor 1α promoter (hEF1a promoter) to drive its expression. The resulting vector was named PCCL-hEF1a-MHC001. The multiple cloning site (MCS) design of this vector allows for the insertion of a nucleic acid fragment containing the Kozak sequence (GCCACCATGG (SEQ ID NO:17)), which is operatively ligated to a nucleic acid sequence encoding a granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) signal peptide (SEQ ID NO:25) located at the N-terminus of the VHH fragment to promote the correct translation, processing, and cell surface expression of the chimeric antigen receptor. The nucleic acid sequences of the aforementioned signal peptide and VHH fragment were designed and inserted upstream of the PCCL-hEF1a-MHC001 vector, and operatively linked to the CAR backbone sequence.

[0131] To construct a single-specific TACI-CAR with a single VHH domain, the nucleic acid sequence encoding the VHH domain was operatively ligated to the 3' end of the nucleic acid sequence encoding the GM-CSFRα signal peptide, resulting in a complete fusion nucleic acid sequence obtained through chemical synthesis. The obtained nucleic acid fragment was treated with EcoRI (5'-GAATTC-3') and SpeI (5'-ACTAGT-3') restriction enzymes, and then cloned into the PCCL-hEF1a-MHC001 backbone vector using molecular cloning techniques known in the art, thereby obtaining a recombinant lentiviral expression vector encoding a monovalent VHH-TACI chimeric antigen receptor. This construction system allows for rapid replacement and modular assembly of different VHH fragments, facilitating subsequent functional screening and in vitro expression validation. The antigen recognition end of the single-target NB38-TACI CAR is the anti-TACI nanobody NB38 obtained in this invention; for comparison, BCMA CARs and BCMA / TACI dual-target CARs can also be constructed. The dual-target CAR contains a tandem BCMA recognition domain and a TACI recognition domain, linked by a linker peptide. The schematic diagrams of the three CAR structures constructed in this embodiment are as follows: Figure 9 As shown. In Figure 9 In the CAR structure shown, the amino acid sequence of BCMA-VHH is shown in SEQ ID NO:26, and the corresponding nucleotide sequence is shown in SEQ ID NO:27. The amino acid sequence of the GS linker is shown in SEQ ID NO:28, and the corresponding nucleotide sequence is shown in SEQ ID NO:29.

[0132] Packaging TACI-related CAR lentivirus: This embodiment uses a 4-plasmid system to package lentivirus: The helper packaging plasmid is pMD2.G: carrying the envelope protein required for lentiviral vector transduction into cells, derived from the VSVG gene of vesicular stomatitis virus. pMDLg / pRRE: carrying the gag gene encoding HIV structural proteins and the Pol gene encoding proteases, reverse transcriptases, and integrases. pRSV-Rev: carrying the REV protein encoding HIV-1 virus, which can improve the stability of mRNA containing RRE elements and promote the transport of mRNA from the nucleus to the cytoplasm.

[0133] A mixture of lentiviral packaging plasmids (containing pMD2.G, pMDLg / pRRE, and pRSV-Rev) was premixed with the vector PCCL-hEF1a-MHC001 containing the VHH fragment (in a pre-optimized ratio with polyetherimide (PEI)) and incubated at room temperature for 5 min. The transfection mixture was then added dropwise to HEK293T cells and gently mixed. The cells were then incubated overnight at 37°C in a 5% CO2 cell incubator. After centrifugation at 500g for 10 min at 4°C, the supernatant was collected and filtered through a 0.45 μm PES filter. Purification was performed using the Lenti QTM purification process, with the following steps: concentration ultrafiltration → affinity chromatography → anion exchange chromatography → concentration → sterile filtration (0.22 μm sterile filtration) → aliquoting.

[0134] (1) TFF Concentration: Ultrafiltration was performed using Cytiva hollow fiber columns UFP-750-E-3×2MA to obtain a high yield while removing a large number of small molecule impurities and improving purity. (2) Core700 Affinity Chromatography: Core700 packing material consists of core-shell polymer microspheres. The shell layer is a neutral hydrophilic layer that does not specifically adsorb proteins. The shell pore size is smaller than the core pore size, and the shell can prevent proteins with a molecular weight greater than 690 kDa from entering the core layer. The core layer is a functional layer for anion exchange and hydrophobic interaction, which can quickly capture protein molecules with a molecular weight less than 690 kDa. (3) Q-column Anion Chromatography: Although the lentiviral vector purity after affinity chromatography is very high, it still contains a large number of empty shell vectors, which do not have the ability to deliver the vector and are prone to causing cytotoxicity. However, since the difference between the empty vector and the intact virus is very small, it is difficult to remove them in the process. After comparative optimization, Q-column chromatography was selected for purification. The empty vector of the vector decreased significantly, and the A260 / 280 value increased, indicating that the empty vector decreased significantly. (4) Storage: The ion exchange chromatography solution was appropriately diluted, filtered through a 0.2 μm membrane, dispensed, and stored at -80 °C.

[0135] Viral transduction titer assay: HEK293T cells were infected with a certain amount of lentivirus. After 72 hours, DNA was extracted to detect the copy number of the target sequence integrated into the host cells (qPCR method), and the transduction titer was calculated. Specific implementation steps: HEK293T cell plating infection (Day 1): HEK293T cells were diluted to 3×10⁻⁶ cells / cells. 5Add 1 mL of diluted cells to each well of a 12-well plate, mix and dilute the sample, and then infect the cells. Set up positive and negative controls simultaneously. After mixing, incubate in a CO2 incubator for 20 ± 2 h. Post-infection medium change (Day 2): Remove the 12-well plate and place it in a biosafety cabinet. Discard the supernatant, gently add 2 mL of fresh culture medium, and continue culturing in a cell culture incubator. Genome extraction (Day 3): Collect cells and extract the genome from HEK293T cells for later use. Transduction titer detection: Prepare standards and qPCR reaction solution, add samples, and perform qPCR detection.

[0136] Calculation of results: Transduction titer (TU mL⁻¹) = (C × N × D × 1000) / V

[0137] C: Average number of viral copies transduced per genome (GAG copy number / RP copy number);

[0138] N: The number of cells at the time of infection;

[0139] D: Dilution factor of the viral vector;

[0140] V: The volume of diluted virus added;

[0141] Contamination rate % = NTC concentration value divided by sample concentration value.

[0142] Preparation of human peripheral blood PBMCs and activation and expansion of T cells: Using a disposable vacuum blood collection needle assembly, 20 mL of blood was aseptically collected from healthy adult volunteers and placed in a blood collection tube containing EDTA anticoagulant. The EDTA-anticoagulated blood was transferred to a 50 mL centrifuge tube, and an equal volume of RPMI-1640 medium was added and gently mixed. The anticoagulated blood was slowly added at a 1:1 ratio to a 15 mL centrifuge tube pre-added with lymphocyte separation medium, and centrifuged at 800×g for 15 min. The white membrane layer was gently aspirated and added to a centrifuge tube containing 10 mL of serum-free RPMI 1640 medium, centrifuged at 500×g for 10 min, and washed twice. The cell pellet was resuspended in 10 mL of serum-free RPMI 1640 medium and centrifuged at 400×g for 8 min. The cell pellet was then resuspended in 5 mL of RPMI-1640 complete medium (10% FBS, 200 IU / mL). In rhIL-2, after trypan blue staining and counting, the concentration was adjusted to 1~3×10⁻⁶. 6 Cell suspensions of cells / ml were prepared for subsequent experiments. PBMCs from healthy donors were subjected to CD3 / CD28 co-stimulation (1×10⁻⁶ cells / ml) using T Cell TransAct (Miltenyi, 130111160) according to the manufacturer's instructions. 6PBMCs were activated and expanded using a 10 μL TransAct agent, and αβT cells were obtained after 7 to 10 days of culture. PBMCs were also activated and expanded using a γδTCR-specific agonist (CN115991774A), and γδT cells were obtained after 10 to 14 days of culture.

[0143] In vitro cytotoxicity assay: To evaluate the specific killing activity of transduced T cells, an in vitro cytotoxicity assay based on a luciferase reporter system was used.

[0144] T-cell transduction and preparation: Pre-activated human peripheral blood T cells were transduced with lentivirus in a solution of 10 μg / mL protamine sulfate, and centrifuged at 37 °C and 1200g for 1 hour to promote viral entry. The cells were then transferred to a 37 °C, 5% CO2 incubator for further culture to induce transgene expression. On day 7 post-transduction, CAR-expressing T cells were collected for CAR transduction efficiency and functional assays. To further evaluate the transduction fit and expression efficiency of the VHH recognition domain CAR construct described in this invention, an EGFP reporter gene was linked to the CAR sequence via P2A, and lentivirus transduction was performed using activated and expanded human αβT cells. Flow cytometry was used to detect the proportion of EGFP-positive cells to assess the transduction and expression efficiency of different CAR constructs. Furthermore, to evaluate the in vitro antitumor activity in the preferred embodiment of this invention, transduced γδT cells were used for functional verification.

[0145] Target cell preparation: Target cells were human multiple myeloma cell lines RPMI-8226-Luc, MM.1S-Luc, and U266-Luc. RPMI-8226 and MM.1S were TACI / BCMA high-expression cell lines, while U266 was a TACI low-expression / BCMA high-expression cell line. All cells were stably transfected with the firefly luciferase gene for real-time monitoring of viable cell count.

[0146] Co-culture and cytotoxicity assay: Effector cells (CAR-T) and target cells were seeded in white 96-well plates at effector-to-target ratios (E:T) of 20:1, 10:1, 5:1, and 1:1 and incubated for 24 hours. After incubation, luciferase substrate was added to the wells, and the luminescence intensity (RLU) reflected the survival rate of target cells. Wherein: RLU maximum (RLU_max): wells containing only target cells (no T cells), representing complete target cell survival; RLU minimum (RLU_min): wells where 1% Triton X-100 was added to lyse target cells, representing complete lysis background; RLU sample (RLU_sample): value measured in the experimental wells. The specific cytotoxicity calculation formula is as follows: Specific cytotoxicity % = 100% * (1 - (RLU sample - RLU minimum) / (RLU maximum - RLU minimum)).

[0147] Cytotoxic cytokine assay: Supernatants from in vitro co-culture assays were collected to assess CAR-induced cytokine release, such as interferon-γ, tumor necrosis factor-α, interleukin-2, and granzyme B (i.e., IFNγ, TNFα, IL2, and GrB) release, detected using ELISA kits. Human IFN-γ one-step ELISA kit (S0C3005), human TNF-α one-step ELISA kit (S0C3024), human IL2 one-step ELISA kit (S0C3001), and human granzyme B one-step ELISA kit (S0C3041) were used according to the manufacturer's instructions to measure IFN-γ, TNF-α, IL2, and GrB levels, respectively. All samples were analyzed in duplicate.

[0148] The results are as follows Figure 10 As shown, NB38-TACI CAR, BCMA CAR, and BCMA / TACI dual-target CAR were successfully constructed. The untransduced T cell group showed extremely low EGFP positive signal, with a positive rate of only 0.25%, indicating a low detection background. In contrast, the NB38-TACI CAR transduction group showed a significant EGFP-positive cell population, with a positive rate of 68.7%; the BCMA CAR transduction group had an EGFP positive rate of 74.4%; and the BCMA / TACI dual-target CAR transduction group had an EGFP positive rate of 61.6%. These results indicate that the three CARs constructed in this invention can achieve high levels of expression in activated and expanded αβT cells. The NB38-TACI single-target CAR and BCMA / TACI dual-target CAR both maintained a transduction positivity rate of over 60%, suggesting that the CAR construct based on the VHH recognition domain has good lentiviral transduction adaptability and expression feasibility.

[0149] Further analysis revealed that the EGFP positivity rate of the dual-target CAR construct was slightly lower than that of the two single-target CARs, which is speculated to be related to the increased overall sequence length, vector load, and expression complexity of the dual-target construct; however, its overall transduction efficiency remained at a high level, sufficient to meet the needs of subsequent functional verification. The NB38-TACI CAR also exhibited high transduction efficiency in single-target construction, indicating that the anti-TACI nanobody NB38 described in this invention not only has good antigen recognition ability but is also suitable for constructing CAR molecules and stably expressing them in T cells.

[0150] In addition, the in vitro killing activity of NB38-TACI CAR, BCMA CAR and BCMA / TACI dual-target CAR transduced γδT cells against multiple myeloma cells constructed in this embodiment is as follows: Figure 11 As shown, all three CAR-γδT cell types exhibited effector-target ratio-dependent cytotoxic activity against multiple myeloma cells, and their overall activity was higher than that of the untransduced γδT cell control group. NB38-TACI CAR-γδT showed strong in vitro cytotoxic activity against RPMI-8226 and MM1S cells, suggesting that the anti-TACI nanobody NB38 described in this invention can effectively mediate the recognition and lysis of TACI-highly expressing target cells. The dual-target CAR-γδT also showed high levels of cytotoxicity in the aforementioned cells. Since U266 cells showed low TACI expression and high BCMA expression, the cytotoxic activity of BCMA CAR-γδT and the dual-target CAR-γδT was higher than that of NB38-TACI CAR-γδT.

[0151] The cytokine detection results were consistent with the in vitro cytotoxicity results, as shown in the figure below. Figure 12 The results show the cytokine release assays after co-culturing different CAR-γδTs with multiple myeloma cells. Compared to the untransduced γδT group, NB38-TACI CAR-γδT induced higher levels of IFN-γ, TNF-α, IL-2, and GrB release after co-culturing with RPMI-8226 and MM1S. In the U266 co-culture system, NB38-TACI CAR-γδT still induced some effector cytokine release, but the overall levels were lower than those of BCMA CAR-γδT and dual-target CAR-γδT.

[0152] The results of this embodiment demonstrate that the NB38-TACICAR-γδT constructed based on the anti-TACI nanobody NB38 of this invention can effectively recognize and kill multiple myeloma cells with high TACI expression and induce significant release of effector cytokines, proving that NB38 can be successfully applied to CAR molecule construction and mediate a clear in vitro antitumor effect. Furthermore, the BCMA / TACI dual-target CAR exhibits broader coverage under different antigen expression backgrounds, suggesting that the TACI targeting strategy can not only be used as an independent treatment regimen but also as a supplement and extension to BCMA-targeted therapy.

[0153] Example 7: In vivo antitumor activity of exemplary TACI-NB38 CAR-γδT in a multiple myeloma xenograft model

[0154] This embodiment aims to evaluate the in vivo antitumor activity of NB38-TACI CAR-γδT cells constructed based on the anti-TACI nanobody NB38 of the present invention in a multiple myeloma xenograft model, and further compare them with untransduced γδT cells, BCMA / TACI dual-target CAR-γδT cells, and BCMA / TACI dual-target CAR-γδT cells to verify the feasibility of the TACI targeting strategy described in this invention in vivo and its application value in targeted therapy of multiple myeloma.

[0155] 1. Sources of laboratory animals and models

[0156] The experimental animals used were NOG mice. NOG mice (NOD.Cg-Prkdc-SCID - Il2rg - / -, abbreviated as NOG) are a highly immunodeficient mouse type, constructed from a NOD (Non-Obese Diabetic) background combined with two gene mutations (Prkdc-SCID and Il2rg - / -). The Prkdc gene mutation leads to the loss of function of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), thereby blocking antigen receptor gene rearrangement during V(D)J recombination between mature T cells and B cells, resulting in the typical SCID (Severe Combined Immunodeficiency) phenotype. The Il2rg gene encodes the common γ chain of multiple cytokine receptors (including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21), a key component essential for the transduction of various cytokines and the promotion of lymphocyte (T, B, NK) and other leukocyte development and maturation. Knockout of the Il2rg gene further weakens immune system function in the SCID background, leading to NK cell deficiency. Therefore, NOG mice simultaneously lack functional T cells, B cells, and NK cells, and exhibit high tolerance to human cell engraftment and tumor xenografting, making them suitable for human tumor models and evaluation of immunotherapy.

[0157] 2. Establishment of a xenotransplantation model for multiple myeloma

[0158] NOG mice aged 6–8 weeks were selected and injected with 2.5 × 10⁻⁶ RPMI-8226-Luc cells via the tail vein. 6 A systemic xenograft model of human multiple myeloma was established using RPMI-8226-Luc cells. These cells stably expressed luciferase and could be used for subsequent bioluminescence imaging (BLI) to dynamically monitor tumor burden in mice. After a certain period following tumor inoculation and once the model was established, the animals were randomly grouped according to the intensity of tumor bioluminescence.

[0159] 3. Dosing grouping and treatment method

[0160] After the model was established, the tumor-bearing mice were divided into the following groups (n=5 per group):

[0161] PBS control group; untransduced γδT cells group (Naive γδT); NB38-TACI CAR-γδT treatment group (TACI-γδT); BCMA CAR-γδT treatment group (BCMA-γδT); BCMA / TACI dual-target CAR-γδT treatment group (BT-γδT). Reference Figure 13The timeline shown is based on day 1 after tumor inoculation. On day 10, each treatment group received the corresponding cell preparation via tail vein infusion, with each dose being 1×10⁻⁶. 7 One cell per animal; the PBS group received an equal volume of PBS, administered 4 days apart, for a total of 3 treatments. In vivo bioluminescence imaging was performed at predetermined time points after drug administration to dynamically observe tumor progression and the tumor-suppressing effects of different treatment groups.

[0162] 4. Tumor burden monitoring

[0163] Reference Figure 13 The timeline shown indicates that in vivo bioluminescence imaging was performed on mice on days 7 (baseline imaging), 21, and 28, respectively. Changes in tumor burden in vivo were evaluated by recording and analyzing the average radiance. The PBS group served as a disease progression control, and the untransduced γδT group served as a background control for cell therapy, comparing the actual antitumor efficacy of the NB38-TACI CAR-γδT described in this invention in vivo.

[0164] 5. Experimental Results

[0165] In vivo bioluminescence imaging results as follows Figure 14 As shown, the tumor burden in mice across all groups was similar at baseline. With prolonged observation, the bioluminescent signal in the PBS group continuously increased, indicating rapid tumor progression in vivo. While the untransduced γδT group showed some tumor suppression compared to the PBS group, the overall tumor burden continued to increase, suggesting that the in vivo anti-tumor effect of natural γδT cells in the model was limited. In contrast, the NB38-TACI CAR-γδT treatment group showed significantly lower bioluminescent signals than the PBS and untransduced γδT groups from the mid-to-late stages of treatment, indicating that this cell preparation could effectively inhibit the growth of RPMI-8226-Luc tumor burden in vivo. At subsequent observation points, the NB38-TACI CAR-γδT group maintained a lower average luminescence intensity level, demonstrating relatively clear and sustained in vivo anti-tumor activity.

[0166] In this model, the BCMA CAR-γδT group and the BCMA / TACI dual-target CAR-γδT group exhibited different degrees of tumor-suppressive effects. The dual-target CAR-γδT group showed the lowest overall tumor bioluminescence signal, suggesting that the dual-target design helps to further enhance in vivo tumor control. The BCMA CAR-γδT group showed certain in vivo activity. Since RPMI-8226 cells simultaneously express TACI and BCMA, the above results indicate that in this double-positive model, the TACI targeting strategy has a clear anti-tumor effect and can complement BCMA targeting.

[0167] The trend of average luminous intensity variation was analyzed, and the results are as follows: Figure 15 As shown, from the mid-to-late stage after treatment, the tumor burden in the NB38-TACI CAR-γδT group was consistently lower than that in the PBS group and the untransduced γδT group, demonstrating that the TACI-targeted CAR-γδT cells constructed based on NB38 described in this invention can effectively recognize and inhibit the growth of TACI-positive multiple myeloma cells in vivo.

[0168] 6. Results and Conclusions

[0169] The results of this embodiment demonstrate that NB38-TACI CAR-γδT cells constructed based on the anti-TACI nanobody NB38 of this invention exhibit clear in vivo anti-tumor efficacy in a NOG mouse model of multiple myeloma xenograft. Compared with the PBS group and the untransduced γδT group, NB38-TACI CAR-γδT significantly reduced tumor bioluminescent signals and delayed tumor progression in vivo, indicating that it can serve as an effective cell therapy strategy targeting TACI. Furthermore, in a tumor model simultaneously expressing BCMA and TACI, BCMA / TACI dual-target CAR-γδT showed a stronger tumor control trend, suggesting that the TACI targeting strategy can not only be used as an independent treatment but also in combination with BCMA targeting, providing a new candidate strategy to address the heterogeneity of multiple myeloma antigen expression and the limitations of BCMA-related treatments.

[0170] Example 8: Preparation and in vitro function of an exemplary NB38-TACI bispecific T cell connector

[0171] This embodiment aims to construct a bispecific T cell engager (TCE) capable of recruiting and activating γδT cells while recognizing the multiple myeloma-associated antigen TACI, and to evaluate its ability to promote γδT cell proliferation and mediate tumor cell killing in vitro.

[0172] 1. Molecular design and construction of γδTCE

[0173] The T cell binding end of the TCE molecule adopts the humanized anti-human γδTCR antibody domain, which is derived from CN115991774A and is used to bind and recruit γδT cells; the tumor recognition end adopts the anti-TACI nanobody NB38 domain obtained by screening in this invention. The two are linked by a flexible linker peptide (e.g., 3×GGGGS (SEQ ID NO:30)) and the Fc region is retained to improve molecular stability, expression level and in vivo half-life.

[0174] To compare the effects of different molecular geometries on the function of dual-specific molecules, three TCE molecules with different structural arrangements were designed, denoted as γδTCE-Ab1, γδTCE-Ab2, and γδTCE-Ab3, respectively. Their designed structures are shown below. Figure 16 As shown (the nucleotide and amino acid sequences of the huMHCc(H2L2) light and heavy chains are shown in CN115991774A, and the huMHCc(H2L2)scfv is formed by linking the variable regions of the huMHCc(H2L2) heavy and light chains via 3×GGGGS). All three conformations contain γδTCR-binding and tumor antigen-binding ends, but their arrangement, linking order, and linking strategies differ. For parallel comparison, the NB38-TACI recognition arm can be replaced with a BCMA recognition arm on the same platform to obtain the corresponding BCMA-TCE molecules, which can be used to evaluate the functional differences between different tumor targets and different conformations.

[0175] The coding sequences of the aforementioned bispecific molecules were cloned into the eukaryotic expression vector pcDNA3.4 and transfected into HEK293T cells for transient expression. The culture supernatant was purified by Protein A affinity chromatography to obtain the corresponding TCE fusion protein. SDS-PAGE analysis showed... Figure 17 The results showed that the purified product in the non-reduced state mainly exhibited a single target band with a purity greater than 90%, and the constructed TCE molecule could be stably expressed and successfully purified.

[0176] 2. In vitro amplification and functional detection

[0177] Human peripheral blood mononuclear cells (PBMCs) were used as the starting cell source. PBMCs were co-cultured with corresponding TCE molecules to evaluate their ability to promote γδT cell proliferation. γδTCE-Ab1, γδTCE-Ab2, or γδTCE-Ab3 were added to the PBMC culture system for cultivation, with a reference γδTCR antibody activation group serving as a control. After 10 days of culture, the proportion of γδT cells was detected by flow cytometry, and exhaustion-related phenotypes such as LAG3, TIM3, and PD1 were further detected. γδT cells expanded by different TCE molecules for 10 days were collected, and total RNA was extracted for transcriptome sequencing analysis. Standard differential expression analysis was used to compare the overall transcriptional characteristics differences among different TCE treatment groups to assess the impact of different TCE conformations on the basal transcriptional program of γδT cells. A PBMC-tumor cell-γδTCE co-culture system was established using RPMI-8226-Luc cells as target cells to evaluate TCE-mediated bridging killing function.

[0178] 3. Experimental Results

[0179] The results are as follows Figure 18As shown, all three TCE molecules can effectively induce or enrich γδT cell expansion in PBMCs. Compared with the reference γδTCR agonist huMHCc (H2L2) derived from CN115991774A, treatment with γδTCE-Ab1, γδTCE-Ab2, and γδTCE-Ab3 all resulted in a more pronounced γδT cell expansion population, indicating that the constructed bispecific molecules can effectively recruit and activate γδT cells in PBMCs. Further detection of exhaustion-related molecules such as LAG3, TIM3, and PD1 (see...) Figure 18 It can be seen that although the expanded γδT cells showed some activation-related phenotypic changes, they did not show an abnormal increase to a significant loss of function, suggesting that the γδT cells in this expansion system still maintained an acceptable functional phenotype.

[0180] The antitumor effect of TCE was further evaluated in a co-culture system of PBMC and RPMI-8226. The results are as follows: Figure 19 As shown, the addition of TCE resulted in an increase in the proportion of γδT cells and a decrease in the proportion of RPMI-8226 target cells in the culture system, indicating that the constructed TCE can effectively bridge γδT cells with TACI-positive tumor cells and induce cytotoxicity. Combined with the aforementioned results, this demonstrates that the NB38-TACI TCE described in this invention can not only be used to recruit and expand γδT cells, but also further mediate their specific killing of multiple myeloma cells.

[0181] RNA sequencing analysis of γδT cells obtained by TCE amplification showed no significant differences in the overall transcriptional program among γδT cells obtained by different TCE amplification methods, indicating that this type of molecule can achieve effective recruitment and activation of γδT cells without significantly altering their basal state.

[0182] 4. Results and Conclusions

[0183] The results of this embodiment demonstrate that the NB38-TACI γδTCE constructed based on the anti-TACI nanobody NB38 of this invention can promote the expansion of PBMC-derived γδT cells and mediate their bridging killing of TACI-positive multiple myeloma cells, exhibiting good in vitro functional activity. This result indicates that NB38 can not only be applied to CAR-γδT cell construction but also further to the design of γδT cell connector molecules, thereby forming multiple cell immunotherapy strategies with TACI as the core target. As a preferred embodiment, the γδTCE platform can also be replaced with a BCMA recognition arm, or the recruitment and anti-tumor effects of γδT cells can be further optimized through different conformations.

[0184] Through the above embodiments, this invention successfully obtained multiple nanobodies targeting human TACI, among which NB38 exhibited high affinity, strong blocking ability, and good specificity in vitro. These nanobodies can not only effectively block BAFF / APRIL-TACI signaling, thereby inhibiting B cell activation and plasma cell differentiation, but also serve as therapeutic target modules for multiple myeloma or B-cell lymphoma, used to construct various therapeutic modalities such as TACI-CAR-T and TACI-TCE, and can be further expanded into drug-conjugated forms. Experimental results show that the nanobodies of this invention have good stability and engineering potential, providing a reliable foundation for developing next-generation precision immunotherapies targeting TACI.

[0185] To address the potential issues of antigen expression heterogeneity and therapeutic limitations in BCMA-targeted therapy for multiple myeloma, this invention provides a nanobody targeting TACI and its CAR application, which can serve as an independent targeting strategy and can further complement the BCMA strategy to expand the coverage of multiple myeloma cells.

[0186] The above detailed description further illustrates the purpose, technical solution, and beneficial effects of the invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nanobody targeting TACI or its antigen-binding fragment, characterized in that, The nanobody or its antigen-binding fragment satisfies one of the following conditions: (1) Contains the amino acid sequence shown in SEQ ID NO:1; (2) Consists of the amino acid sequence shown in SEQ ID NO:1; and (3) Contains an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:1, and contains amino acid sequences such as CDR1 shown in SEQ ID NO:18, CDR2 shown in SEQ ID NO:19 and CDR3 shown in SEQ ID NO:

20.

2. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the nanobody or its antigen-binding fragment as described in claim 1.

3. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 2 or expresses the nanobody or its antigen-binding fragment of claim 1.

4. A host cell, characterized in that, The host cell contains the nucleic acid molecule of claim 2 or the expression vector of claim 3.

5. A fusion protein, characterized in that, The fusion protein comprises the nanobody or its antigen-binding fragment as described in claim 1.

6. The fusion protein according to claim 5, characterized in that, The fusion protein further comprises one or more of a long-acting peptide and a tag sequence linked to the nanobody or its antigen-binding fragment.

7. The fusion protein according to claim 5, characterized in that, The fusion protein further comprises one or more of an Fc domain and an albumin-binding domain linked to the nanobody or its antigen-binding fragment, wherein the Fc domain is derived from human IgG1, IgG2, IgG4 or engineered variants thereof.

8. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor includes an antigen recognition domain, which comprises the nanobody or its antigen-binding fragment as described in claim 1.

9. The chimeric antigen receptor according to claim 8, characterized in that, The chimeric antigen receptor comprises, from N-terminus to C-terminus, a signal peptide, an antigen recognition domain, a hinge region and / or a spacer region, a transmembrane region, and an intracellular signal transduction domain.

10. The chimeric antigen receptor according to claim 9, characterized in that, The intracellular signal transduction domain includes CD3ζ and further includes one or more co-stimulatory domains selected from CD28, 4-1BB, OX40, ICOS, DAP10, and DAP12.

11. The chimeric antigen receptor according to any one of claims 8-10, characterized in that, The chimeric antigen receptor further includes a second antigen recognition domain that targets BCMA.

12. An immune effector cell, characterized in that, The immune effector cells express the chimeric antigen receptor as described in any one of claims 8-11.

13. The immune effector cell according to claim 12, characterized in that, The immune effector cells are selected from one or more of the following: peripheral blood T cells, NK cells, iPSC-derived immune cells, macrophages, and dendritic cells.

14. The immune effector cell according to claim 12, characterized in that, The immune effector cells are selected from one or more of the following: γδT cells, αβT cells, and NKT cells.

15. The immune effector cell according to claim 12, characterized in that, The immune effector cells are cytotoxic T lymphocytes.

16. A bispecific or multispecific cell connector, characterized in that, The cell connector includes: (1) A tumor recognition end comprising the nanobody or its antigen-binding fragment as described in claim 1; and (2) Immune cell binding end, which can bind to molecules on the surface of immune effector cells.

17. The cell connector according to claim 16, characterized in that, The immune cell binding terminal can bind to γδTCR, CD3, CD16, NKp30, NKp46, NKG2D or combinations thereof.

18. The cell connector according to claim 16 or 17, characterized in that, The cell connector further includes a second tumor recognition end that targets BCMA.

19. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises one or more of the nanobody or antigen-binding fragment of claim 1, the fusion protein of any one of claims 5-7, the chimeric antigen receptor of any one of claims 8-11, the immune effector cell of any one of claims 12-15, and the cell connector of any one of claims 16-18, as well as a pharmaceutically acceptable carrier or excipient.

20. Use of the nanobody of claim 1 or its antigen-binding fragment, the nucleic acid molecule of claim 2, the expression vector of claim 3, the host cell of claim 4, the fusion protein of any one of claims 5-7, the chimeric antigen receptor of any one of claims 8-11, the immune effector cell of any one of claims 12-15, the cell connector of any one of claims 16-18, or the pharmaceutical composition of claim 19 in the preparation of a medicament for treating TACI-positive diseases; The TACI-positive disease mentioned above is multiple myeloma.

21. The use according to claim 20, characterized in that, The drug is used in combination with or sequentially with proteasome inhibitors, immunomodulators, chemotherapy drugs, radiotherapy, or combinations thereof.

22. The use according to claim 21, characterized in that, The immunomodulator is selected from one or more of the following: BCMA-targeting drugs and monoclonal antibodies.

23. The use according to claim 22, characterized in that, The BCMA-targeting drug is selected from one or more of the following: BCMA-CAR and BCMA-TCE.

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