Anti-human slamf7 antibody, genes and uses thereof

Abstract

The application belongs to the technical field of biotechnology, and discloses an anti-human SLAMF7 antibody, genes and application thereof. The amino acid sequence of the SLAMF7 antibody is shown as SEQ ID NO. 1-10. The anti-human SLAMF7 nanobody of some examples of the application has good affinity to SLAMF7. The CAR-T cell constructed by taking the nanobody as an antigen recognition domain can efficiently recognize and kill tumor cells expressing SLAMF7, and exhibits significant anti-tumor activity, and is expected to bring more effective and safer treatment options for multiple myeloma patients.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an anti-human SLAMF7 antibody, gene, and its applications. Background Technology

[0002] Signaling lymphocytic activation molecule family 7 (SLAMF7), also known as CD319, CRACC, and CS1, is a transmembrane protein receptor with a relative molecular weight of 66 kDa, belonging to the type I transmembrane glycoprotein of the immunoglobulin CD2 superfamily. Its extracellular domain contains an immunoglobulin variable domain (IgV) and an immunoglobulin constant 2 (C2)-like domain. The IgV-like domain lacks typical disulfide bonds, exhibiting strong self-affinity and the ability to bind ligands or antibodies. In contrast, the C2-like domain contains two disulfide bonds, which helps maintain the structural stability of the SLAMF receptor. The intracellular domain contains immune receptor tyrosine-based switch motifs (ITSMs), which, upon tyrosine phosphorylation, can recruit single lymphocyte activation signaling molecule-binding protein (SLAM-associated protein, SAP) or Ewing's sarcoma-associated transcript 2 (EAT-2) to initiate downstream signal transduction (Li J, et al. Mol Cancer.2025;24(1):145.).

[0003] SLAMF7 is expressed on the surface of various immune cells, including natural killer (NK) cells, cytotoxic T lymphocytes, mature dendritic cells, activated B cells, and monocytes. In NK cells, SLAMF7 participates in tumor recognition and clearance by binding to CRACC on the surface of target cells. This process promotes the binding of EAT-2 (an adaptor protein containing an SH2 domain) within NK cells to phosphorylated ITSM of SLAMF7, thereby activating tyrosine phosphorylation of phospholipase C-γ (PLC-γ) and phosphatidylinositol 3-kinase (PI3K) (Gutierrez-Guerrero A, et al. Eur J Immunol. 2022;52(1):62-74.). The PLC-γ pathway drives NK cell degranulation, releasing cytotoxic substances; the PI3K pathway promotes the production of cytokines (such as IL-2, IL-15, and IL-12) through the Akt / mTOR axis, further enhancing the degranulation response. The presence of EAT-2 is crucial for this pathway; its absence inhibits the killing function of NK cells, thereby weakening anti-tumor immunity and potentially promoting the progression of diseases such as multiple myeloma (Chu E, et al. Curr Oncol. 2023;30(9):7891-7903.). In T cells, activation of SLAMF7 leads to phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT3. Unlike NK cells, this phosphorylation does not enhance anti-tumor activity; instead, it induces the expression of various inhibitory receptors (such as PD-1) and transcription factors associated with T cell exhaustion, thereby inhibiting CD8⁺ T cell function and helping tumors evade immune attack (O'Connell P, et al. J Immunol.2021;206(1):193-205.).

[0004] SLAMF7 is highly expressed in multiple myeloma (MM) cells and participates in the development and progression of the disease. In the bone marrow microenvironment, MM cells interact with the extracellular matrix (ECM) and bone marrow stromal cells (BMSCs), activating signaling pathways such as MAPK, Notch, and PI3K, thereby evading immune surveillance and promoting tumor growth (Ho M, et al. Curr Oncol. 2022;29(11):8975-9005.). SLAMF7 exists in two forms: membrane-bound and soluble (sSLAMF7). The expression level of membrane-bound SLAMF7 is not significantly associated with MM disease progression, while sSLAMF7 may reflect disease progression. sSLAMF7 can be detected in the serum of approximately 31% of MM patients, but not in healthy individuals (Ishibashi M, et al. Oncotarget. 2018;9(78):34784-34793.). Patients positive for sSLAMF7 typically exhibit more aggressive clinical features, shorter progression-free survival, and a higher proportion of them in stage III patients of the Revised International Stages System (R-ISS). Therefore, sSLAMF7 holds promise as a potential prognostic biomarker. sSLAMF7 can bind to membrane-bound SLAMF7 on the surface of MM cells, thereby activating the SHP-2 and ERK signaling pathways. Immunofluorescence staining showed that this binding process is accompanied by phosphorylation activation of SHP-2 and ERK, and induces ERK nuclear translocation. However, these signals did not trigger an anti-tumor effect; instead, they promoted tumor cell proliferation through ERK kinase, thereby accelerating MM progression (Kikuchi J, et al. Leukemia. 2020;34(1):180-195.).

[0005] Currently, there are two main strategies for treating MM with SLAMF7: elotuzumab and CAR-T cell therapy.

[0006] Elotuzumab (trade name Empliciti®) is a humanized IgG1κ monoclonal antibody that was approved by the US FDA in 2015 for the treatment of adult multiple myeloma (MM). This drug exerts its antitumor effect through a dual mechanism: firstly, its Fab fragment binds to SLAMF7 on the surface of MM cells, and its Fc fragment binds to FcγRIIIa / CD16 on the surface of NK cells, inducing antibody-dependent cell-mediated cytotoxicity (ADCC) (Taniwaki M, et al. Mediterr J Hematol Infect Dis. 2018;10(1):e2018014.); secondly, it directly activates NK cells, promoting the secretion of IL-2 and TNF-α, thereby enhancing granzyme B release and increasing the NK cell's ability to kill MM cells (Pazina T, et al. Cancer Immunol Res. 2019;7(10):1633-1646.).

[0007] Soluble SLAMF7 (sSLAMF7) can serve as a biomarker for predicting the efficacy of evotuzumab, and its high expression is associated with better treatment response. Elotuzumab monotherapy has limited efficacy and requires combination therapy with other drugs. In relapsed / refractory patients, its combination with lenalidomide / dexamethasone or pomalidomide / dexamethasone has been shown to significantly prolong progression-free survival and improve response rates (Dimopoulos MA, et al. N Engl J Med. 2018;379(19):1811-1822.). In newly diagnosed patients, the combination regimen has also shown good induction and consolidation therapy effects, but its advantage in improving survival in patients unsuitable for autologous stem cell transplantation (ASCT) remains unclear. Furthermore, combination regimens of evotuzumab with proteasome inhibitors such as bortezomib and carfilzomib also show potential. Currently, the combination of this drug with novel drugs (such as belantatumab malfodotin and celinotox) and its sequential or combination strategies with CD38 monoclonal antibodies are still being explored.

[0008] Chimeric antigen receptor T-cell (CAR-T) therapy targeting SLAMF7 is an immunotherapy for multiple myeloma. It involves genetically engineering T cells from a patient or donor to express a receptor that recognizes the SLAMF7 antigen. Once activated, these CAR-T cells proliferate and release cytokines, directly killing SLAMF7-positive tumor cells. Compared to monoclonal antibodies (such as ileotuzumab), CAR-T therapy has greater potency and a different mechanism of action. Its advantages include the ability to recognize and eliminate tumor cells at lower antigen densities and the capacity for self-proliferation. However, this also brings higher risks, including more severe side effects (such as cytokine release syndrome) and off-target toxicity to normal SLAMF7-positive cells (such as NK cells, some T cells, and B cells), potentially leading to an increased risk of infection. To address these challenges, researchers are developing various strategies. For example, induced suicide genes (such as caspase-9-based systems) have been integrated into CAR-T cells to eliminate CAR-T cells in cases of severe toxicity, thereby protecting normal immune cells (Amatya C, et al. Mol Ther. 2021;29(2):702-717.). In addition, CAR-T products using virus-free transposon systems (such as SleepingBeauty) and "universal" allogeneic CAR-T cells (UCARTCS1) prepared using gene editing technologies (such as TALEN) have emerged, reducing the risk of graft-versus-host disease and cannibalism by knocking out endogenous T cell receptors and the SLAMF7 gene (Teoh PJ, et al. Blood Cancer J. 2021;11(4):84.). A more promising strategy is to develop bispecific CAR-T cells that simultaneously target SLAMF7 and BCMA, aiming to expand tumor coverage and reduce tumor escape due to antigen loss (Moares N, et al. Front Immunol. 2025;16:1613222.). Currently, several early-stage clinical trials of SLAMF7 CAR-T are underway to evaluate its safety and preliminary efficacy in patients with relapsed / refractory multiple myeloma and to explore optimal dosing. Summary of the Invention

[0009] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide an anti-human SLAMF7 antibody, gene and its application.

[0010] The technical solution adopted in this invention is: A first aspect of the present invention provides: an anti-human SLAMF7 nanobody, comprising a framework region and a complementarity-determining region, the nanobody being capable of specifically binding to human SLAMF7, wherein: CDR1, CDR2, and CDR3 are selected from the CDR combinations corresponding to the nanobodies shown in SEQ ID NO. 1 to SEQ ID NO. 10, or, the CDR1, CDR2, and CDR3 have at least 85%, 90%, or 95% sequence identity with the CDR combinations corresponding to the nanobodies shown in SEQ ID NO. 1 to SEQ ID NO. 10, and maintain the ability to specifically bind to human SLAMF7.

[0011] In some instances, based on the IMGT numbering scheme, the amino acid sequences of the antibody complementarity-determining regions CDR1–CDR3 are as follows: P1-A4, the amino acid sequences of CDR1 to CDR3 are: GSFLSLDS, FSRRGYT, NKANWQPNMYE; P2-A6, the amino acid sequences of its CDR1 to CDR3 are: GSFLSLDS, FRRGST, NKFNWQPIMYE; P1-B7, the amino acid sequences of CDR1 to CDR3 are: GSFLSLDS, ITRRGYT, NKANWQPNMYE; P1-A3, the amino acid sequences of its CDR1 to CDR3 are: GSFLSLDS, FIRGYR, NKGNWQPTMYE; P1-A12, the amino acid sequences of its CDR1 to CDR3 are: GSFLSLDS, IIRGYR, NKGNWQPTMYE; P1-F6, the amino acid sequences of CDR1 to CDR3 are: GFTFSSYD, INTGGGST, ARGDPFSDSDRY; P1-B12, the amino acid sequences of its CDR1 to CDR3 are: GFTFSSYD, INTGGDST, ARGDPFSDSPT; P1-H1, the amino acid sequences of its CDR1 to CDR3 are: GFTFSSFD, IKTGGGNT, ATGRPWGGSSASI; P2-A2, the amino acid sequences of its CDR1 to CDR3 are: GFTFSRYG, ISSTGGIT, NGNQAFRSVYNY; P1-G11 has the following amino acid sequences for CDR1 to CDR3: DSFDRHI, INFSDGRT, AAEPHDMDRGTLLLIESNFNS.

[0012] In some instances, the amino acid sequence is as shown in any one of SEQ ID NO.1 to SEQ ID NO.10; or it is a functional variant of the above-mentioned nanobody obtained by humanization, chimerism, Fc fusion, affinity optimization, half-life extension or other protein engineering modifications; wherein the functional variant retains the ability to specifically bind to human SLAMF7.

[0013] A second aspect of the invention provides: a bivalent or multivalent antibody comprising the complementarity-determining region of the nanobody described in the first aspect of the invention and capable of specifically binding to human SLAMF7 molecules.

[0014] A third aspect of the invention provides: a gene encoding an antibody described in the first or second aspect of the invention.

[0015] A fourth aspect of the present invention provides: an expression system containing the gene described in the third aspect of the present invention and expressing the antibody described in the first or second aspect of the present invention.

[0016] In some instances, the expression system includes, but is not limited to, expression vectors, host cells, or recombinant expression systems containing the gene.

[0017] A fifth aspect of the invention provides: the application of the antibody described in the first or second aspect of the invention, the application comprising: To prepare therapeutic antibody drugs targeting SLAMF7; Preparation of antibody-drug conjugates targeting SLAMF7; Prepare or modify a drug delivery vehicle to target cells expressing SLAMF7; Construct chimeric antigen receptor (CAR) immune cells targeting SLAMF7; Prepare diagnostic reagents or immunoadsorbents that target SLAMF7.

[0018] In some instances, the conjugates in the antibody-drug conjugates include, but are not limited to, cytotoxic drugs, tracers, radionuclides, immunomodulatory molecules, or other functional molecules.

[0019] In some instances, the CAR includes an SLAMF7 binding domain, a linker, a transmembrane domain, and an intracellular signaling domain, wherein the SLAMF7 binding domain includes the nanobody described in the first aspect of the present invention.

[0020] In some instances, the intracellular signaling domain includes CD3ζ and at least one co-stimulatory domain selected from CD28, 4-1BB, OX40, and ICOS.

[0021] In some instances, the linker is selected from one of (G4S)n, (EAAAK)n, Whitlow 218 linker, LHL, and IgG hinge regions.

[0022] In some instances, the CAR immune cells are CAR-T cells, CAR-NK cells, or other genetically engineered immune cells.

[0023] These features can be combined arbitrarily as long as they do not conflict with each other.

[0024] A sixth aspect of the present invention provides the use of the nanobody described in the first aspect of the present invention, the bivalent or multivalent antibody described in the second aspect of the present invention, the gene described in the third aspect of the present invention, the expression system described in the fourth aspect of the present invention, and the CAR immune cell described in the fifth aspect of the present invention in the preparation of a drug for the prevention or treatment of SLAMF7 positive tumors.

[0025] In some instances, the SLAMF7-positive tumors include multiple myeloma and other hematologic malignancies that express SLAMF7.

[0026] The beneficial effects of this invention are: The anti-human SLAMF7 nanobodies of some examples of the present invention have a good affinity for SLAMF7. CAR-T cells constructed with these nanobodies as antigen recognition domains can efficiently recognize and kill tumor cells expressing SLAMF7, exhibiting significant anti-tumor activity, and are expected to bring more effective and safer treatment options to patients with multiple myeloma. Attached Figure Description

[0027] Figure 1 The interaction sites of 10 nanobodies with different sequences with SLAMF7(ECD) were predicted using AlphaFold 3.0 (grey: SLAMF7(ECD), red: nanobodies).

[0028] Figure 2 Ten nanobodies with different sequences were subjected to ELISA on bacteriophages.

[0029] Figure 3 Schematic diagram of Anti-SLAMF7 CAR-T structure.

[0030] Figure 4 The changing trend of CAR expression levels in Anti-SLAMF7 CAR-T cells during culture.

[0031] Figure 5 Proliferation of Anti-SLAMF7 CAR-T cells.

[0032] Figure 6 Changes in SLAMF7 expression in Anti-SLAMF7 CAR-T cells.

[0033] Figure 7 Experimental results on the binding ability of Anti-SLAMF7 CAR-T cells to SLAMF7 antigen.

[0034] Figure 8 Results of anti-SLAMF7 CAR-T cells killing tumor cells with different SLAMF7 expression levels. Detailed Implementation

[0035] SLAMF7 is an important target for immunotherapy of multiple myeloma, but it still faces challenges such as drug resistance and limited therapeutic efficacy in clinical practice. To address these limitations, this invention focuses on developing novel nanobodies targeting SLAMF7. Nanobodies have advantages such as small molecular weight, high affinity, structural stability, and good tissue penetration. Using alpaca immunization-binding phage display technology, the inventors successfully screened a series of novel nanobodies that specifically recognize SLAMF7. Using the screened high-affinity nanobodies as antigen recognition domains, a SLAMF7-targeting chimeric antigen receptor was constructed. In vitro functional experiments confirmed that this CAR-T cell can efficiently recognize and kill tumor cells expressing SLAMF7, exhibiting significant anti-tumor activity, and is expected to provide more effective and safer treatment options for patients with multiple myeloma.

[0036] The technical solution of the present invention will be further illustrated below with examples. Example 1: Preparation of SLAMF7 (ECD) antigen

[0037] Construction and extraction of pCAGGS-SLAMF7 ECD-IgG1 Fc plasmid: The SLAMF7 ECD-IgG1 Fc gene fragment was synthesized by the company and constructed into the pCAGGS vector using molecular cloning methods. After successful plasmid sequencing, a high concentration of endotoxin-free plasmid was obtained according to the OMEGAEndo-Free Plasmid Midi Kit D6915 procedure. After quality control by agarose gel electrophoresis, it was stored at -20℃ for later use.

[0038] Obtaining SLAMF7 (ECD) antigen: Mix 5 ml Opti-MEM with 50 μg pCAGGS-SLAMF7 ECD-IgG1 Fc plasmid, then add 125 μl PEI 40K and slowly invert to mix. Incubate at room temperature in the dark for 30 min, then slowly add to a solution with a density of 3.0 × 10⁻⁶. 6Cells were cultured at a density of 293F cells / ml on a shaker for 72 hours. The cell supernatant was collected by centrifugation, resuspended in fresh culture medium, and cultured for another 48 hours. The cells were then centrifuged again, and the supernatant was retained. Both supernatants were centrifuged at high speed to remove cell debris, filtered through a 0.45µm filter, and stored on ice. The cell supernatant was slowly passed through a Protein G column using a peristaltic pump. After washing with 20 column volumes of binding buffer, protein was eluted with elution buffer. The pH was adjusted with neutralization buffer, and the concentration was measured using a nanodropper. Following the Solarbio Thrombin instructions, the protein was dialyzed to prepare a buffer solution suitable for enzyme digestion. An appropriate amount of Thrombin was added, and the solution was incubated at room temperature for 16 hours. SDS-PAGE was used to assess the digestion effect. The digested protein sample was then passed through a Protein G column again to remove IgG1 Fc protein, yielding the SLAMF7 (ECD) antigen. SDS-PAGE analysis showed a protein purity greater than 90%. ELISA assays confirmed that the expressed and purified SLAMF7 (ECD) could bind to the commercially available anti-SLAMF7 specific antibody in a dose-dependent manner, and the signal was significantly higher than that of the negative control, indicating that the expressed protein maintained its native conformation and possessed the correct antigenicity and biological activity. Example 2: Immunization of alpacas using SLAMF7 (ECD) antigen

[0039] The SLAMF7 (ECD) antigen was emulsified and then used to immunize alpacas. Immunization was performed every two weeks for a total of six immunizations. Blood samples were collected before and after each immunization for serum titer determination (ELISA). After the sixth immunization, the titer reached 10. 5 At a certain level, database building and screening can be performed. Example 3: RNA extraction and reverse transcription

[0040] RNA extraction: Peripheral blood lymphocytes preserved with Trizol were transferred to 1.5 ml centrifuge tubes, and 1 / 5 volume of chloroform was added and mixed. After standing at room temperature for 5 minutes, the mixture was centrifuged at 12000g for 15 minutes at 4°C. The supernatant was carefully transferred to a new centrifuge tube, and an equal volume of isopropanol was added. After standing at room temperature, the mixture was centrifuged again. The precipitate in each tube was washed with 1 ml of 75% ethanol, centrifuged to remove the ethanol, dried, and dissolved in an appropriate amount of RNase-free water. All samples were combined to obtain the total RNA extracted.

[0041] Reverse transcription of cDNA: The total RNA obtained was reverse transcribed using the Takara reverse transcription kit. The total RNA sample was divided into two portions. One portion was transcribed into cDNA using the Oligo dT Primer provided in the kit, and the other portion was transcribed into cDNA using the Random 6-mers provided in the kit, following the instructions of the reverse transcription kit. The cDNA samples were then stored in two separate centrifuge tubes. Example 4: PCR amplification

[0042] First round of PCR amplification: The first round of PCR reaction was performed using cDNA as a template. PCR amplification was carried out using Taq DNA Polymerase Hot Start enzyme. The PCR reaction system was prepared according to the table below: cDNA 4μl 1st-For / 1st-Rev 2μl / 2μl dNTP Mix 4μl 10×ExTaq Buffer 5μl HS Ex Taq 0.25μl <![CDATA[ddH2O]]> Up to 50μl After the reaction, all PCR products were subjected to 1% agarose gel electrophoresis, and the target fragment bands with a size of approximately 600 bp were excised and recovered. All purified and recovered products from the first round of PCR were collected into a centrifuge tube, which is the first round of PCR amplification product, and stored at -20℃.

[0043] Second round of PCR amplification: Use the products from the first round of PCR amplification as templates for the second round of PCR reaction. Configure the PCR reaction system according to the table below: First-round PCR product recovery 0.2μl 2nd-For / 2nd-Rev 2μl / 2μl dNTP Mix 4μl 10×ExTaq Buffer 5μl HS Ex Taq 0.25μl <![CDATA[ddH2O]]> Up to 50μl After the reaction, the DNA in the PCR reaction solution was purified using a universal DNA purification and recovery kit. The recovered product was collected into a centrifuge tube, which is the second round of PCR amplification product. At the same time, 2 μl of the recovered product was measured and recorded using a nucleic acid concentration meter, and the remaining product was stored at -20℃. Example 5: Enzyme digestion and ligation

[0044] Vector and PCR product digestion: pComb3XSS was used as the phage plasmid vector. 10 μg of pComb3XSS vector and 6 μg of the second-round PCR amplification product were digested with restriction endonucleases SpeI and SacI, respectively, and incubated at 37°C for 4 h. The pComb3XSS vector and the second-round PCR amplification product were purified using a DNA recovery and purification kit and stored at 4°C.

[0045] Ligation: The vector and fragment are ligated. The ligation reaction system is prepared according to the table below: PCR products 1.6μg Vector (pComb3XSS) 4μg T4 ligase 30μl 10×T4 reaction buffer 200μl <![CDATA[ddH2O]]> Up to 2000μl The ligation reaction was incubated overnight (approximately 16 hours) at 4°C. The ligation reaction solution was purified using a universal DNA purification and recovery kit. The concentration of the recovered product was measured and stored at 4°C. Example 6: Construction of bacterial and bacteriophage libraries

[0046] Bacterial library construction: After electroporation using TG1 competent cells, the cells were thawed at 37°C for 1 hour. 100 μl of the thawed cells were then serially diluted 10-fold and plated. The cells were incubated overnight at 37°C. All remaining bacterial cultures were collected and evenly spread onto 150 mm circular culture plates (2×YT containing 100 μg / ml Amp, 2% glucose, and 2% agarose) and incubated inverted at 37°C overnight. The number of transformed colonies obtainable from all reactions was calculated based on the dilution factor and single colony count; this number represents the library capacity. Simultaneously, 48 single clones were randomly selected from the serially diluted plates for colony PCR to verify the clone positivity rate of the bacterial library. Colonies from the overnight-incubated 150 mm circular culture plates were scraped off using 2×YT liquid medium, placed in 50 ml centrifuge tubes, and their OD600 value was measured. A final concentration of 20% glycerol was added, and the tubes were stored at -80°C.

[0047] Phage library preparation: The bacterial library was inoculated into a medium containing antibiotics and cultured until the OD600 reached 0.5-0.55. Based on the helper phage titer, helper phages were added at a ratio of 1:20 (bacteria:phages), and incubated at 37°C and 250 rpm for 30-60 min. A final concentration of 50 μg / ml Kana was added, and the mixture was incubated overnight. The overnight culture was centrifuged, and the supernatant was added to 1 / 4 volume of pre-chilled PEG / NaCl stock solution. The mixture was thoroughly mixed, incubated on ice for 30 min, centrifuged, and the supernatant was discarded. The supernatant was resuspended in 1 ml of PBS, centrifuged again, and the mixture was centrifuged once more. Another 1 / 4 volume of pre-chilled PEG / NaCl stock solution was added to the supernatant, the mixture was thoroughly mixed, incubated on ice, centrifuged, and the supernatant was discarded. The supernatant was resuspended in PBS and stored at -80°C. This yielded the purified phage library. At this point, the phage library titer was 2.4 × 10⁻⁶. 13 pfu / ml.

[0048] Positive clone rate colony PCR: The colony PCR agarose gel electrophoresis results showed that 46 out of the 48 selected single clones were positive clones, with a positive rate of 96%, which met the requirements for the positive clone rate of the bacterial library.

[0049] Sequence diversity: 48 single clones were randomly selected and sequenced, and then translated into amino acid sequences using GENtle software. Sequence diversity comparison showed that all 48 sequences were independent sequences, with 100% diversity, and the bacterial library diversity met the requirements. Example 7: Phage library screening based on immunotube immobilized antigens

[0050] First round of screening: Coat the immunotubes with antigen and incubate overnight at 4°C by slow rotation. Discard the liquid in the overnight coated immunotubes, add 2 ml of PBS buffer and wash the immunotubes three times at room temperature, rotating for 5 min each time. Add 2 ml of blocking buffer (3% BSA) and incubate at room temperature for 1 h by rotation. First, discard the blocking buffer in the negative sieve immunotubes, wash, and add phage as the input for the first round of screening phage library. After incubation at room temperature for 1 h by rotation, transfer the incubated liquid from the negative sieve immunotubes to the positive sieve immunotubes and continue incubation. After incubation, add PBST buffer and wash the immunotubes 20 times at room temperature, rotating for 5 min each time. Discard the liquid in the immunotubes, removing as much residual liquid as possible, add 1 ml of 0.25 mg / ml Trypsin solution, and elute by rotation at room temperature for 30 min. Add 10 μl of 10% AEBSF to stop elution, and transfer the solution in the immunotubes to a new 1.5 ml centrifuge tube, which is the phage elution buffer for the first round of screening.

[0051] First-round phage eluent titer assay: The first-round phage eluent was serially diluted 10 times. TG1 bacterial suspension was added to each dilution, and the mixture was incubated at 37°C for 30 min. Then, it was added dropwise to a solid culture medium containing antibiotics and incubated upside down overnight. Single colonies on the plate were counted, and the phage library titer was calculated.

[0052] Amplification of the first round of phage elution: The phage elution obtained after the first round of screening was added to TG1 bacterial culture and cultured. All bacterial culture was evenly spread onto solid plates and incubated overnight. The colonies from the overnight-cultured square plates were gently scraped off with a spreading stick and collected; this is the amplified bacterial sub-library. The preparation of the first round of phage sub-library is similar to the phage library preparation method in Example 6, with helper phages added to the first-round bacterial library and incubated overnight. The overnight-cultured bacterial culture was precipitated with PEG / 2.5M NaCl solution and resuspended in PBS to obtain the phage sub-library, and library titer was simultaneously determined. The second and subsequent rounds of screening were similar to the first round, with more stringent screening conditions as the number of rounds increased, ultimately obtaining phage clones with high affinity for the SLAMF7 (ECD) antigen. Example 8: Monoclonal ELISA Detection

[0053] The phage eluates from the second and third rounds of screening were serially diluted, and TG1 was added to each well for further incubation. The bacterial culture was evenly spread onto solid culture plates containing antibiotics and incubated overnight. Single colonies were randomly picked from the overnight culture plates and cultured in sterile 96-well cell culture plates (P1-P2 plates were picked from the second round, P3-P4 plates from the third round). Helper phages were added to each well and the plates were incubated overnight. After overnight incubation, the phages were obtained by centrifugation. The plates were coated overnight with the positive and negative screening antigens SLAMF7 (ECD) and hIgG Fc, respectively, followed by washing with 200 µl of PBS buffer per well. 200 µl of blocking buffer (3% BSA) was added to each well and the plates were blocked at room temperature for 1 h. The blocking buffer was discarded, and 200 µl of PBST buffer was added to each well. The plates were washed three times at room temperature for 10 min each time. The centrifuged phages were added to each well and the plates were incubated at room temperature for 2 h. The liquid in the plates was discarded, and 200 µl of PBST buffer was added to each well for washing. Add 100 μl of M13 Bacteriophage Antibody (HRP) and Mouse Mab diluted 1:8000 in blocking buffer to each well and incubate at room temperature for 1 h. After washing, add 100 µl of TMB single-component chromogenic solution to each well and incubate in the dark for 2-3 min. Stop the reaction by adding 100 µl of 1M HCl to each well. Read the OD450 value using a microplate reader, record and save the data. To rule out false positives, clones initially identified as positive were subjected to a second ELISA verification using the same method. Clones that tested positive in both ELISA tests were sequenced. The sequenced sequences were analyzed using GENtle software for sequence alignment, and the antibody sequences were translated into amino acids using GENtle software. Example 9: Prediction and structural analysis of nanobody binding epitopes

[0054] For the obtained nanobodies, AlphaFold 3.0 was used to predict their interaction regions with the SLAMF7 (ECD) antigen. Based on their enrichment during the screening process, 10 clones targeting different epitopes of the antigen were selected (e.g., ...). Figure 1 (As shown). Subsequently, ELISA was performed on bacteriophages, using the same method as in Example 8. The results showed that all 10 clones exhibited high affinity for the SLAMF7 antigen, while showing no significant binding to irrelevant antigens such as hIgG Fc and BSA. This indicates that the screened nanobodies possess excellent targeting and affinity. Figure 2 ).

[0055] The amino acid sequences of the 10 nanobodies are as follows: P1-A4 amino acid sequence: DVQLQESGGGLVQPGESLRLSCAASGSFLSLDSMGWYRQSPGHPRELIASFSRRGYTYYDDAVKGRFTISRDNADNIWYLQMNSLKPEDTAVYHCNKANWQPNMYEWGQGTQVTVSS (SEQ ID NO.1).

[0056] P2-A6 amino acid sequence: DVQLQESGGGLVQPGESLRLSCAASGSFLSLDSMGWYRQPPGHPRELVASFRRGSTYYDDAVKGRFTISRDNADNIWYLQMNSLKPEDTAVYYCNKFNWQPIMYEWGQGTQVTVSS (SEQ ID NO.2).

[0057] P1-B7 amino acid sequence: DVQLQESGGGLVQPGESLRLSCAASGSFLSLDSMGWYRQPPGHPRELVASITRRGYTFYDDAVKGRFTISRDNADNIWYLQMNSLKPEDTAVYHCNKANWQPNMYEWGQGTQVTVSS (SEQ ID NO.3).

[0058] P1-A3 amino acid sequence: DVQLQESGGGLVQPGESLRLSCAASGSFLSLDSMGWYRQPPGHPRELVASFIRGYRYYDDAVKGRFTISRDNADNIWYLQMNSLKPEDTGVYYCNKGNWQPTMYEWGQGTQVTVSS (SEQ ID NO.4).

[0059] P1-A12 amino acid sequence: DVQLQESGGGLVQPGESLRLSCAASGSFLSLDSMGWYRQPPGHPRELVASIIRGYRYYDDAVKGRFTISRDNADNIWYLQMNSLKPEDTAVYYCNKGNWQPTMYEWGQGTQVTVSS (SEQ ID NO.5).

[0060] P1-F6 amino acid sequence: DVQLQESGGGMVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGPEWISFINTGGGSTYYDDSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCARGDPFSDSDRYWGQGTQVTVSS (SEQ ID NO. 6).

[0061] P1-B12 amino acid sequence: DVQLQESGGGMVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGPEWISFINTGGDSTYYDDSVKGRFTISRDNAKNTLYLQMKSLKPEDTALYTCARGDPFSDSPTWGQGTQVTVSS (SEQ ID NO. 7).

[0062] P1-H1 amino acid sequence: DVQLQESGGGMVQPGGSLRLSCVASGFTFSSFDMSWVRQAPGKGPEWISFIKTGGGNTYYDDSVKGRFTISRDDAKNTLYLQMNSLKPEDTALYYCATGRPWGGSSASIRGRGTQVTVSS (SEQ ID NO. 8).

[0063] P2-A2 amino acid sequence: DVQLQESGGGLVQPGESLRLSCAASGFTFSRYGMSWYRQAPGKERELVAGISSTGGITKYKDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAAYYCNGNQAFRSVYNYWGQGTQVTVSS (SEQ ID NO. 9).

[0064] P1-G11 amino acid sequence: DVQLQESGGGLVQPGGSLRLSCAASDSFDRHIIGWFRQAPGKEREGVSSINFSDGRTYYDSDVKGRFIISRDNAKNMVYLQMSSLKPEDTAVYYCAAEPHDMDRGTLLLIESNFNSWGQGTQVTVSS (SEQ ID NO. 10).

[0065] The CDR analysis results of each nanobody based on the IMGT numbering scheme are shown in Table 1 below.

[0066] Table 1. CDR analysis results of different nanobodies P1-A4 GSFLSLDS FSRRGYT NKANWQPNMYE P2-A6 GSFLSLDS FRRGST NKFNWQPIMYE P1-B7 GSFLSLDS ITRRGYT NKANWQPNMYE P1-A3 GSFLSLDS FIRGYR NKGNWQPTMYE P1-A12 GSFLSLDS IIRGYR NKGNWQPTMYE P1-F6 GFTFSSYD INTGGGST ARGDPFSDSDRY P1-B12 GFTFSSYD INTGGDST ARGDPFSDSPT P1-H1 GFTFSSFD IKTGGGNT ATGRPWGGSSASI P2-A2 GFTFSRYG ISSTGGIT NGNQAFRSVYNY P1-G11 DSFDRHI INFSDGRT AAEPHDMDRGTLLLIESNFNS The sequence numbers of each CDR sequence are shown in Table 2.

[0067] Table 2. CDR sequence numbers of different nanobodies P1-A4 11 18 16 P2-A6 11 12 13 P1-B7 11 15 16 P1-A3 11 24 22 P1-A12 11 26 22 P1-F6 17 30 27 P1-B12 17 28 25 P1-H1 14 21 19 P2-A2 23 33 31 P1-G11 20 32 29 The complementarity-determining region (CDR) sequences of the aforementioned 10 nanobodies are highly homologous, their core antigen-binding motifs are highly conserved, and they all recognize the same target antigen. Specifically: The first homologous subgroup (P1-A4, P2-A6, P1-B7, P1-A3, and P1-A12): The CDR1 sequences of the five antibodies in this subgroup are completely identical (GSFLSLDS), with no amino acid mutations, directly proving that they share the same germline V gene rearrangement background and originated from the same initial B cell clone. The CDR2 region is 6-7 amino acids in length, sharing the conserved RG core motif, with only 1-2 amino acid point mutations (such as phenylalanine F→isoleucine I, serine S→threonine T or isoleucine I, tyrosine Y→serine S, threonine T→arginine R, etc.). These mutation sites are all located at the N-terminal edge of the CDR loop or in the non-core contact region. The physicochemical properties of the amino acids before and after the substitution are highly similar, and the overall folding and antigen binding pattern of the loop region are not changed. The CDR3 region is 11 amino acids long and shares the conserved sequence backbone of NK-X-NWQP-X-MYE. Only a few amino acid substitutions exist at non-core antigen contact sites (such as alanine A → phenylalanine F or glycine G, asparagine N → isoleucine I or threonine T). The core functional residues that directly participate in antigen recognition are completely conserved.

[0068] 1. CDR2, position 1: Phenylalanine (F) → Isoleucine (I) Both are nonpolar hydrophobic amino acids with large hydrophobic surface areas in their side chains and similar levels of steric hindrance. This site is located at the junction of the N-terminus of CDR2 and the backbone region, and does not directly participate in the core contact of the antigen epitope; the substitution only fine-tunes the local hydrophobic interaction strength and does not change the overall turn conformation of the CDR ring, which is the most classic type of conserved hydrophobic amino acid substitution in antibody affinity maturation.

[0069] 2. CDR2, position 2: serine (S) → threonine (T) or isoleucine (I) Serine and threonine are both polar hydroxyl amino acids, with hydroxyl groups on their side chains that can participate in hydrogen bond formation. The only difference is the slight length of a single methyl group on the side chain. This is the most classic conserved substitution type in the high-frequency mutations of antibodies in somatic cells, and it hardly changes the steric hindrance and chemical properties of the site. When the mutation at this site is isoleucine, it is located in the non-core antigen contact region at the edge of the CDR ring. It only slightly adjusts the local hydrophobic environment and does not destroy the core spatial conformation of the CDR2 ring or the antigen recognition specificity.

[0070] 3. CDR2 core site, GR dipeptide motif Arginine (R) is a positively charged basic polar amino acid and a core functional residue mediating antigen-antibody electrostatic interactions and hydrogen bonding. Glycine (G) is the amino acid with the smallest side chain and a key structural residue maintaining the CDR ring-turn conformation and conformational flexibility. This core functional motif is completely conserved in all antibodies, directly demonstrating that the antigen-binding mode of each antibody and the CDR2 core spatial backbone have not undergone essential changes, serving as a core sequence marker that they originate from the same B cell clone.

[0071] 4. At position 6 of CDR2, tyrosine (Y) → serine (S) Both have hydroxyl functional groups on their side chains and belong to the polar amino acids that can participate in hydrogen bond formation. Only tyrosine has an additional aromatic ring hydrophobic skeleton. When replaced by serine, it only reduces the steric hindrance of the side chain and weakens the local hydrophobic effect. The core hydroxyl bonding ability and polar characteristics are completely preserved. It is a semi-conservative substitution between polar hydroxyl amino acids and does not change the overall conformation of the CDR2 ring and the core properties of antigen binding.

[0072] 5. CDR2 C-terminus, threonine (T) → arginine (R) Threonine is a polar hydroxy amino acid, and arginine is a basic polar amino acid. Both are polar amino acids, and their side chains can form hydrogen bonds. This is a fine-tuning substitution of charge within polar amino acids. This site is close to the backbone region and does not directly participate in antigen binding, but only fine-tunes the local charge distribution.

[0073] 6. At position 3 of CDR3, alanine (A) → phenylalanine (F) or glycine (G) Phenylalanine, alanine, and glycine are all nonpolar hydrophobic amino acids, with decreasing hydrophobicity and steric hindrance in that order, representing a progressively conserved substitution within the hydrophobic amino acid family. This site is located on the lateral side of the CDR3 ring apex, and the substitution only slightly adjusts the steric hindrance and hydrophobic interaction strength at the ring apex. The introduction of glycine can slightly increase the flexibility of the ring region, a common mutation for optimizing antibody binding kinetics, without altering the properties of the CDR3 core binding interface.

[0074] 7. At position 8 of CDR3, asparagine (N) → isoleucine (I) or threonine (T) This site is located on the lateral edge of the CDR3 loop and is not a core functional site that directly contacts the antigen epitope. Asparagine and threonine are both short-chain polar amino acids, and their side chains can form hydrogen bonds through polar groups. Their steric hindrance is highly similar, which is a classic type of conserved substitution in protein evolution, hardly changing the local chemical microenvironment. The mutation of asparagine to isoleucine only adjusts the local polar environment to a hydrophobic environment. Since the site does not participate in the recognition of the core antigen, it only fine-tunes the local hydrophobic properties of the loop region and does not destroy the overall spatial conformation of CDR3 and the antigen binding specificity. These are all routine conserved property adjustments in the same clonal affinity maturation process.

[0075] The second homologous subgroup (P1-F6, P1-B12, P1-H1, P2-A2): The CDR1 region of the four antibodies in this subgroup is 8 amino acids in length, sharing the core sequence motif of GFTFS-XXX, with sequence homology exceeding 60%. The mutation sites are all amino acid substitutions with similar physicochemical properties (such as serine S→arginine R, tyrosine Y→phenylalanine F, aspartic acid D→glycine G, etc.), maintaining the hydrophobic core and spatial structure of CDR1; the CDR2 region is 8 amino acids in length, rich in glycine and polar residues, and retains the conserved ring conformation; the CDR3 region is concentrated in length of 11-13 amino acids, with consistent charge distribution characteristics and extended ring structure, and the core functional domain for antigen binding is highly conserved.

[0076] P1-G11 clone: ​​This antibody is a mutant with a longer CDR3 in the same clonal lineage. Its CDR1 and CDR2 still retain the same length characteristics and residue properties as the above subgroups. The sequence extension of CDR3 is the result of somatic insertion mutation and does not change its recognition specificity for the target antigen. It still belongs to the functional variant under the same inventive concept.

[0077] 1. At position 6 of CDR1, serine (S) → arginine (R) Both are polar amino acids, only slightly adjusting the local positive charge, and both side chains can participate in hydrogen bond formation, belonging to semi-conservative substitution between polar amino acids; this site is located at the C-terminal edge of CDR1 and does not directly participate in the binding of the antigen core epitope.

[0078] 2. At position 7 of CDR1, tyrosine (Y) → phenylalanine (F) Both belong to aromatic amino acids, and their side chains are based on a benzene ring as the core backbone, with steric hindrance highly consistent with the main chain conformation. This mutation only removes the phenolic hydroxyl group at the para position of the tyrosine benzene ring, completely preserving the hydrophobic core structure of the aromatic ring. The mutation only reduces one potential hydrogen bond interaction site, without changing the hydrophobic backbone properties or side chain space occupancy of this site. It is a typical conserved amino acid substitution in the antibody CDR region and will not change the overall folding pattern of the CDR1 ring or the antigen recognition specificity.

[0079] 3. At position 8 of CDR1, aspartic acid (D) → glycine (G) The replacement increases the conformational flexibility of the CDR1 C-terminus, which is a local structural fine-tuning; since the site is located at the junction of CDR1 and the backbone region, it does not affect the structure of the core antigen binding region.

[0080] 4. CDR1 differences between P1 and G11 The antibody's CDR1 region has accumulated a large number of high-frequency somatic mutations and its sequence differentiation degree is higher than that of other antibodies in the subgroup. However, it still retains the core aromatic hydrophobic residue (phenylalanine) in this region. Overall, it maintains the residue composition characteristics of "hydrophobic residues as structural support and polar / charged residues interspersed", which can ensure the formation of CDR1 loop. No large-scale sequence insertion, deletion or frame rearrangement has occurred, which is consistent with the somatic mutation evolution characteristics of distant mutants in the same clonal lineage.

[0081] 5. At position 2 of CDR2, asparagine (N) → lysine (K) or serine (S) Asparagine is a polar, uncharged amino acid with an amide group on its side chain. When mutated to lysine, both belong to the long-side-chain polar amino acid family, with similar carbon chain backbone lengths and the ability to form hydrogen bonds at their ends, exhibiting only minor differences in charge properties. This represents a semi-conserved substitution between long-side-chain polar amino acids. When mutated to serine, both belong to the polar amino acid family, with polar functional groups on their side chains that can participate in hydrogen bond interactions. Furthermore, this site is located in the non-core antigen contact region of CDR2, and the shortening of the side chain length will not disrupt the overall folding of the loop region. Neither type of substitution alters the core flexible backbone mediated by the continuous glycine motif of CDR2. These are conserved mutations among the high-frequency mutations in antibody somatic cells and will not change the core specificity of antigen recognition.

[0082] 6. At position 6 of CDR2, glycine (G) → aspartic acid (D) Glycine is the least nonpolar amino acid, while aspartic acid is a short-side-chain acidic polar amino acid. Substitution only introduces negative charge locally in the ring region and increases the ability to form hydrogen bonds. Since glycine itself has no side chain occupancy, the steric hindrance after substitution does not increase significantly. It is a tolerable local property fine-tuning that does not destroy the loop region flexibility mediated by the GG motif.

[0083] 7. At position 7 of CDR2, serine (S) → asparagine (N) or isoleucine (I) Serine and asparagine are both polar, uncharged amino acids with similar side chains capable of hydrogen bonding and similar spatial dimensions. They are the most typical conserved substitution pairs in protein evolution, and the local polar environment and spatial conformation are hardly affected after the substitution. The mutation of serine to isoleucine is a semi-conserved substitution from polar to nonpolar. This site is located at the C-terminal edge region of the CDR2 ring and does not directly participate in the contact of the antigen core epitope. It only slightly adjusts the local hydrophobic environment and does not change the overall folding and core functional conformation of the CDR2 ring.

[0084] 8. CDR2 differences between P1 and G11 The antibody retains the glycine conformation residues, and the overall residue polarity distribution is consistent with that of other antibodies. All mutations are point mutations that accumulate, and no frame rearrangement has occurred.

[0085] 9. P1-F6 and P1-B12 are highly homologous in CDR3, with the only difference being the C-terminal DRY→PT: both tyrosine (Y) and threonine (T) contain hydroxyl groups, which are conservative substitutions of polar hydroxyl amino acids; arginine (R) → proline (P) only increases the rigidity of the C-terminus, which is a local conformational fine-tuning, and the sequences of the core antigen-binding regions of the two are completely identical.

[0086] 10. Using the CDR3 sequences of P1-F6 as a reference, although there is a one-amino acid length difference between P1-H1, key residues such as alanine, proline, and glycine, which maintain the overall conformation of the CDR3 ring, are conserved. The phenylalanine-tryptophan substitution is a classic conserved substitution of aromatic hydrophobic amino acids, and many polar hydroxyl residues are retained. Only a small number of property exchanges exist at non-core sites on the ring edge. The CDR3 lengths of P2-A2 are consistent, and sequence substitutions are mainly conserved / semi-conserved. Aromatic hydrophobic residues and basic charged residues are retained in homologous functional regions. The sequence differentiation of CDR3 in both is a typical manifestation of the gradual accumulation of high-frequency mutations in somatic cells. The hydrophobic, charge, and polar characteristics of the core binding interface are highly similar, and the antigen recognition specificity has not been changed, which is consistent with the evolutionary pattern of the same clonal lineage.

[0087] 11. The long CDR3 of P1-G11 is a product of somatic insertion mutation, which is a common evolutionary way to increase the antigen contact area during antibody affinity maturation; and it still targets the same antigen, belonging to the functionally optimized variant under the same inventive concept. Example 10: Construction of SLAMF7-targeted CAR-T

[0088] Construction and extraction of Anti-SLAMF7(VHH) CAR-T plasmid: The screened nanobody sequences (10 in total) were molecularly cloned and linked to the CD8 hinge-CD8 transmembrane domain-4-1BB co-stimulatory domain-CD3ζ domain to construct a second-generation CAR plasmid targeting SLAMF7. Figure 3After successful sequencing, the endotoxin-free plasmid was obtained by following the instructions of the OMEGA Endo-Free Plasmid Mini Kit D6948. After quality control by agarose gel electrophoresis, it was stored at -20℃ for later use.

[0089] Lentiviral Packaging and Titer Detection: The target plasmid, lentiviral packaging helper plasmid, and Lipofectamine 2000 transfection reagent were added to Opti-MEM and incubated at room temperature in the dark for 30 min. Then, the plasmid was slowly added to 293T cells (with confluence greater than 90%), and the medium was replaced with fresh medium after 6 h. After 48 h, the supernatant was collected, centrifuged to remove cell debris, and the virus was aliquoted and stored at -80℃. The lentivirus was diluted 5, 25, and 125 times, and used to infect 293FT cells. CAR expression on the cells was detected, and the lentiviral titer was calculated.

[0090] Preparation of SLAMF7-targeted CAR-T: PBMCs from healthy donors were rapidly revived in a 37°C water bath after being removed from liquid nitrogen. After centrifugation, cell counts were performed, and the cell density was adjusted to 1 million / ml. CD3 / CD28 magnetic beads and 300 IU / ml IL-2 were added for activation for 24 h. Activated cells were collected by centrifugation, and 1 million T cells, lentivirus (MOI=3), IL-2 (300 IU / ml), and polybrene (6 μg / ml) were added to each well of a 24-well plate. After centrifugation and incubation for 15 h, 1 ml of fresh culture medium was added to each well. On day 6, the medium was changed and the magnetic beads were removed. CAR expression levels were then detected, and the results are as follows: Figure 4 As shown, except for P2-A2 CAR-T cells, the other cells maintained a CAR expression rate of over 75% throughout the culture process, while the CAR expression level of P2-A2 cells gradually decreased with culture time. Regarding CAR-T cell proliferation, all CAR-T cells exhibited strong proliferative capacity. Figure 5 ).

[0091] Since SLAMF7 is also expressed on the surface of T cells, we monitored the dynamic expression of SLAMF7 in T cells during activation and CAR transduction. Figure 6 The results showed that SLAMF7 expression was high on T cells before activation (day 0); its expression decreased after activation and CAR transduction, but gradually recovered with subsequent culture. Example 11: Detection of Anti-SLAMF7 CAR-T binding ability to antigen

[0092] SLAMF7(ECD)-hIgG Fc antigen was serially diluted 10-fold with PBS buffer, resulting in seven concentration gradients. CAR-T cells of adjusted density were added to 100 μl per well of a 96-well U-shaped plate, centrifuged at 1000g for 5 min, and the supernatant was discarded. 100 μl of different antigen concentrations were added to each well to suspend the cells, and the plates were incubated on ice for 1.5 h. The cells were then centrifuged again, the supernatant was discarded, and the cells were washed twice with PBS. Anti-hIgG Fc (PE) flow cytometry antibody was then added, and the plates were incubated on ice in the dark for 1 h. After centrifugation, the cells were washed with PBS to remove unbound antibodies. Finally, the cells were resuspended in 200 μl of PBS for flow cytometry analysis. The results are shown in Table 3. Figure 7 As shown, except for the P2-A2 clone EC 50 Apart from the higher level, the remaining clones of EC 50 All values ​​were below 0.5 nM, indicating that it has extremely strong SLAMF7 antigen binding ability.

[0093] Table 3. Comparison of the binding capacity of Anti-SLAMF7 CAR-T cells to SLAMF7 antigen. P2-A6 0.1 P1-B7 0.28 P1-A4 0.19 P1-H1 0.47 P1-A3 0.07 P1-A12 0.05 P1-B12 0.1 P1-F6 0.06 P1-G11 0.12 P2-A2 2.9 Example 12: Comparison of the killing activity of Anti-SLAMF7 CAR-T tumor cells

[0094] To evaluate the cytotoxic activity of CAR-T cells, tumor cell lines with different SLAMF7 expression levels were selected for experiments: MM.1S (high SLAMF7 expression, 2+), Daudi (low SLAMF7 expression, +), and K562 (SLAMF7 negative, -). First, tumor cells were incubated at 37°C in the dark for 15 minutes with 1×CFSE staining solution prepared in PBS containing 5% FBS, followed by washing three times with RPMI 1640 complete medium and resuspending for counting. After centrifugation and counting, CAR-T cells were co-cultured with tumor cells at an effector-to-target ratio of 1:1.6 in 96-well plates. The cytotoxic effect was detected by flow cytometry after 24 hours. Results are as follows: Figure 8 As shown, in MM.1S cells with high SLAMF7 expression, all CAR-T cells except for the P2-A2 clone exhibited strong specific killing effects; in Daudi cells with low SLAMF7 expression, CAR-T (P1-A3) and CAR-T (P1-A12) showed the best killing effects, while CAR-T (P1-G11) and CAR-T (P2-A2) showed weaker effects; in SLAMF7-negative K562 cells, no obvious non-specific killing effects were observed.

[0095] Combination Figure 1Epitope prediction analysis showed that nanobodies targeting the juxtamembranous region of the SLAMF7 antigen (P1-G11 and P2-A2) had weak binding and killing abilities; while nanobodies targeting the middle region of the antigen (P1-A3 and P1-A12) exhibited higher binding activity and killing efficacy. These results provide a key basis for the optimization and development of SLAMF7-targeting antibodies.

[0096] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A nanobody against human SLAMF7, comprising a framework region and a complementarity-determining region (CDR), characterized in that, The nanobody is capable of specifically binding to human SLAMF7, wherein: CDR1, CDR2 and CDR3 are selected from the CDR combinations corresponding to the nanobodies shown in SEQ ID NO.1 to SEQ ID NO.10, or, the CDR1, CDR2 and CDR3 have at least 85%, 90% or 95% sequence identity with the CDR combinations corresponding to the nanobodies shown in SEQ ID NO.1 to SEQ ID NO.10, and maintain the ability to specifically bind to human SLAMF7.

2. The nanobody according to claim 1, characterized in that, Based on the IMGT numbering scheme, the amino acid sequences of the antibody complementarity-determining regions CDR1–CDR3 are as follows: P1-A4, the amino acid sequences of CDR1 to CDR3 are: GSFLSLDS, FSRRGYT, NKANWQPNMYE; P2-A6, the amino acid sequences of its CDR1 to CDR3 are: GSFLSLDS, FRRGST, NKFNWQPIMYE; P1-B7, the amino acid sequences of CDR1 to CDR3 are: GSFLSLDS, ITRRGYT, NKANWQPNMYE; P1-A3, the amino acid sequences of its CDR1 to CDR3 are: GSFLSLDS, FIRGYR, NKGNWQPTMYE; P1-A12, the amino acid sequences of its CDR1 to CDR3 are: GSFLSLDS, IIRGYR, NKGNWQPTMYE; P1-F6, the amino acid sequences of CDR1 to CDR3 are: GFTFSSYD, INTGGGST, ARGDPFSDSDRY; P1-B12, the amino acid sequences of its CDR1 to CDR3 are: GFTFSSYD, INTGGDST, ARGDPFSDSPT; P1-H1, the amino acid sequences of its CDR1 to CDR3 are: GFTFSSFD, IKTGGGNT, ATGRPWGGSSASI; P2-A2, the amino acid sequences of its CDR1 to CDR3 are: GFTFSRYG, ISSTGGIT, NGNQAFRSVYNY; P1-G11 has the following amino acid sequences for CDR1 to CDR3: DSFDRHI, INFSDGRT, AAEPHDMDRGTLLLIESNFNS.

3. The nanobody according to claim 1 or 2, characterized in that, Its amino acid sequence is shown in any one of SEQ ID NO.1 to SEQ ID NO.10; or it is a functional variant of the above nanobody obtained by humanization, chimerism, Fc fusion, affinity optimization, half-life extension or other protein engineering modifications; wherein the functional variant retains the ability to specifically bind to human SLAMF7.

4. A bivalent or multivalent antibody, characterized in that, It includes two or more nanobody binding domains as described in any one of claims 1-3, and is capable of specifically binding to human SLAMF7 molecules.

5. A gene encoding the antibody according to any one of claims 1 to 4.

6. An expression system, characterized in that, It includes the gene of claim 5 and is capable of expressing the antibody of any one of claims 1 to 4; wherein the expression system includes an expression vector, a host cell, or a recombinant expression system containing the gene.

7. The application of the antibody according to any one of claims 1 to 4, characterized in that, The applications include: To prepare therapeutic antibody drugs targeting SLAMF7; Preparation of antibody-drug conjugates targeting SLAMF7; Prepare or modify a drug delivery vehicle to target cells expressing SLAMF7; Construct chimeric antigen receptor (CAR) immune cells targeting SLAMF7; Prepare diagnostic reagents or immunoadsorbents that target SLAMF7.

8. The application according to claim 7, characterized in that, The antibody-drug conjugate includes cytotoxic drugs, tracers, radionuclides, immunomodulatory molecules, or other functional molecules; and / or, The CAR immune cells are CAR-T cells, CAR-NK cells, or other genetically engineered immune cells; and / or, The CAR includes an SLAMF7 binding domain, a linker, a transmembrane domain, and an intracellular signaling domain. The SLAMF7 binding domain includes the nanobody described in any one of claims 1 to 3.

9. The application according to claim 8, characterized in that, The intracellular signaling domain includes CD3ζ and at least one co-stimulatory domain; wherein the co-stimulatory domain is selected from CD28, 4-1BB, OX40, ICOS or a combination thereof; the linker is selected from one or more of (G4S)n, (EAAAK)n, Whitlow linker, LHL, and IgG hinge region.

10. The use of the antibody according to any one of claims 1 to 4, the gene according to claim 5, the expression system according to claim 6, or the CAR-immune cell according to any one of claims 7 to 9 in the preparation of a drug for the prevention or treatment of SLAMF7-positive tumors; wherein, The SLAMF7-positive tumors include multiple myeloma, plasma cell tumors, and other hematologic malignancies that express SLAMF7.

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