Antigen binding domains that bind the cell surface receptor bcam and uses thereof

Abstract

The application discloses an antigen binding domain combining with a cell surface receptor BCAM and application, and finds that the CNF1 D5 domain is a fragment with specificity and high affinity in combination with BCAM. The CNF1 D5 with only specificity and high affinity in combination with BCAM is used as the antigen binding domain combining with BCAM, high-efficiency specific targeting of BCAM can be realized, potential problems caused by other functions or epitopes of CNF1 are not introduced, and safety is high. In the embodiment of the application, a CNF1 D5 (C866S) mutant with high affinity which is determined to be reserved is used as an antigen recognition module, is fused with an anti-CD3 single-chain antibody (scFv), and a CD3 scFv-CNF1 D5 bispecific molecule is constructed, T cells and tumor cells with high expression of BCAM can be bridged at the same time, and T cells are mediated to kill efficiently and specifically. The application provides a new immunotherapy molecule and cell strategy based on a high-affinity non-antibody scaffold for malignant tumors with high expression of BCAM.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an antigen-binding domain that binds to the cell surface receptor BCAM and its applications. Background Technology

[0002] Immunotherapy, especially cancer immunotherapy represented by immune cell conjugates, has the core advantage of mobilizing the patient's own immune system to attack tumors, which is fundamentally different from traditional treatments (surgery, chemotherapy, and radiotherapy). First, the immune system has a memory function; once activated to recognize cancer cells, some memory T cells can survive for a long time. This may lead to continued tumor control even after drug withdrawal, achieving long-term "survival with the tumor" or even clinical cure. For some patients with advanced melanoma and lung cancer, immunotherapy has created miracles of long-term survival. Second, when immunotherapy takes effect, the activated immune cells not only attack the primary tumor site but may also attack metastatic lesions in other parts of the body via blood circulation. This is particularly significant when radiotherapy is combined with immunotherapy. Third, traditional targeted drugs often develop resistance due to new mutations in the tumor. The immune system can recognize multiple antigens and, theoretically, can simultaneously attack cancer cells of different genotypes and respond to newly emerging mutant strains, reducing the risk of rapid drug resistance. Fourth, the side effects of immunotherapy stem from the overactivation of the immune system (immune-related adverse events, such as rash, colitis, and pneumonia), rather than the direct cytotoxic effects of chemotherapy. Although serious side effects may occur, common symptoms of traditional chemotherapy such as severe nausea, vomiting, hair loss, and bone marrow suppression are very rare, and patients may have a higher quality of life. Fifth, immunotherapy has shown encouraging results for tumors that are insensitive to or resistant to traditional radiotherapy and chemotherapy (such as advanced melanoma, renal cell carcinoma, and Hodgkin's lymphoma). Therefore, there is an urgent need to develop various immunotherapy products targeting different tumor antigens. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing technologies by providing an antigen-binding domain that binds to the cell surface receptor BCAM and its applications.

[0004] In a first aspect, the present invention provides an antigen-binding domain that binds to the cell surface receptor BCAM.

[0005] CNF1 (cytotoxic necrosis factor 1), a protein toxin produced by *E. coli*, is primarily associated with urinary tract infections, intestinal diseases, and meningitis. It activates Rho GTPases within host cells, leading to cytoskeleton rearrangement, multinucleation, and cell necrosis, thereby disrupting the epithelial barrier and facilitating bacterial invasion and infection. CNF1 toxin hijacks the host laminin-511 receptor Lu / BCAM to enter cells. Based on this, this invention experimentally screens and verifies that the CNF1 D5 domain is a fragment with specific and high affinity for binding to BCAM. Fragments with other functions and epitopes, such as CNF1 D1-4, are discarded. Using only CNF1 D5, which specifically and with high affinity for BCAM, as the antigen-binding domain for BCAM, highly efficient and specific targeting of BCAM can be achieved without introducing other safety issues. This provides a novel molecular and cellular strategy for immunotherapy based on a high-affinity non-antibody scaffold for malignant tumors with high BCAM expression.

[0006] The present invention discloses an antigen-binding domain for binding cell surface receptor BCAM, wherein the antigen-binding domain for binding cell surface receptor BCAM is the D5 domain (720-1014 amino acids) of CNF1, and its amino acid sequence is shown in SEQ ID NO.1 or a variant thereof, and its nucleotide sequence is shown in SEQ ID NO.2 or a variant thereof.

[0007] SEQ ID NO.1:

[0008] SIESTSKSNFQKLSRGNIDVLKGRGSISSTRQRAIYPYFEAANADEQQPLFFYIKKDRFDNHGYDQYFYDNTVGLNGIPTLNTYTGEIPSDSSSLGSTYWKKYNLTNETSIIRVSNSARGANGIKIALEEVQEGKPVIITSGNLSGS TTIVARKEGYIYKVHTGTTKSLAGFTSTTGVKKAVEVLELLTKEPIPRVEGIMSNDFLVDYLSENFEDSLITYSSSEKKPDSQITIIRDNVSVFPYFLDNIPEHGFGTSATVLVRVDGNVVVRSLSESYSLNADASEISVLKVFSKKF

[0009] In a second aspect, the present invention provides the use of the antigen-binding domain of the cell surface receptor BCAM in the preparation of an immune product for treating cancer or autoimmune diseases, wherein the immune product is an immune cell conjugate; and one end of the immune cell conjugate that recognizes tumor-associated antigens is the antigen-binding domain of the cell surface receptor BCAM.

[0010] Generally, the term "variant" refers to a molecule that has one or more additions, substitutions (which are generally conserved in nature), and / or deletions of natural sequences relative to the natural molecule, provided that these modifications do not impair its biological activity, and that the variant is "fundamentally homologous" to the reference molecule. Typically, the protein sequence of such variants has a high degree of sequence homology or identity with the reference sequence; for example, when comparing the two sequences, the protein sequence homology or identity is greater than 25%, typically greater than 50% to 70%, and even more specifically 80%, or 85% or higher, such as at least 90%, or 95% or higher.

[0011] Furthermore, the immune cell conjugate is a T cell conjugate, an NK cell conjugate, or a γδ T cell conjugate.

[0012] Furthermore, the T cell conjugate is a bispecific T cell conjugate, wherein the other end recognizes CD3 on T cells.

[0013] Furthermore, the nucleotide sequence that identifies the conjugate portion of CD3 on T cells is shown in SEQ ID NO.4.

[0014] Furthermore, the cancers mentioned are ovarian cancer, liver cancer, lung cancer, stomach cancer, breast cancer, colorectal cancer, prostate cancer, skin cancer, and other cancers that express BCAM (see Int. J. Mol. Sci. 2024, 25, 7268. https: / / doi.org / 10.3390 / ijms25137268; and Discover Oncology 2024 15:38. https: / / doi.org / 10.1007 / s12672-024-01244-1).

[0015] In a third aspect, the present invention provides a bispecific T-cell conjugate, wherein one end of the bispecific T-cell conjugate specifically recognizes BCAM and the other end recognizes CD3 of T cells; wherein the end that specifically recognizes BCAM is the antigen-binding domain that binds to BCAM.

[0016] Furthermore, the nucleotide sequence of CD3 is shown in SEQ ID NO.4.

[0017] In a fourth aspect, the present invention provides a polynucleotide encoding the bispecific T-cell conjugate; the polynucleotide comprises a first nucleic acid and a second nucleic acid; the first nucleic acid has a sequence as described in SEQ ID NO.4 or a variant thereof; and the second nucleic acid has a sequence as described in SEQ ID NO.2 or a variant thereof.

[0018] In a fifth aspect, the present invention provides a recombinant expression vector comprising the polynucleotides described in the fourth aspect.

[0019] In a sixth aspect, the present invention provides a transformant that expresses the bispecific T-cell conjugate as described in the third aspect, or contains the polynucleotide as described in the fourth aspect, or contains the recombinant expression vector as described in the fifth aspect.

[0020] In a seventh aspect, the present invention provides a method for preparing the bispecific T cell conjugate, comprising the following steps:

[0021] (1) Construct a gene vector encoding the CD3 gene that recognizes T cells and the antigen-binding domain of the cell surface receptor BCAM.

[0022] (2) The gene vector encoding step (1) is transfected into mammalian cells, cultured, and then the protein is purified to obtain the bispecific T cell conjugate.

[0023] Preferably, the vector in step (1) is a pCDNA3.1 vector; and the mammalian cell in step (2) is a 293F cell.

[0024] In an eighth aspect, the present invention provides a pharmaceutical composition comprising an immune cell conjugate as described in the first or second aspect, a bispecific T cell conjugate as described in the third aspect, a polynucleotide as described in the fourth aspect, a recombinant expression vector as described in the fifth aspect, and / or a transformant as described in the sixth aspect, and optionally a pharmaceutically acceptable carrier and / or excipient.

[0025] A ninth aspect of the present invention provides the use of an immune cell conjugate as described in the first or second aspect, a bispecific T cell conjugate as described in the third aspect, a polynucleotide as described in the fourth aspect, a recombinant expression vector as described in the fifth aspect, and / or a transformant as described in the sixth aspect, or a pharmaceutical composition as described in the eighth aspect, in the preparation of an antitumor drug or a drug for treating cancer.

[0026] In a tenth aspect of the present invention, a method for treating cancer is provided, wherein the method comprises administering to a subject in need an effective amount of an immune cell conjugate, as described in the third aspect, a bispecific T cell conjugate, a pharmaceutical composition as described in the eighth aspect, or an antitumor drug or a drug for treating cancer as described in the ninth aspect.

[0027] Furthermore, the cancers mentioned are cancers that express BCAM, such as ovarian cancer, liver cancer, lung cancer, stomach cancer, breast cancer, colorectal cancer, prostate cancer, and skin cancer.

[0028] The beneficial effects of this invention are as follows: This invention abandons fragments such as CNF1 D1-4 that have other functions and epitopes, and uses CNF1 D5, which binds specifically and with high affinity to BCAM, as the antigen-binding domain for binding the cell surface receptor BCAM. This allows for highly efficient and specific targeting of BCAM without introducing potential problems caused by other functions or epitopes on CNF1, resulting in high safety. Simultaneously, a CNF1 D5-based T-cell engager (TCE) was designed and constructed. This TCE efficiently guides T cells to kill BCAM-expressing tumor cells in vitro and significantly inhibits tumor progression in a mouse model of metastatic ovarian cancer. Attached Figure Description

[0029] Figure 1 This diagram shows the experimental results of CNF1 D5 C866S binding to BCAM with high affinity. In the diagram, A is a schematic diagram of the CNF1 domain; B is the crystal structure of the CNF1-BCAM complex. CNF1 mainly interacts with BCAM V2 via β26 of D5; C shows the ITC titration results of full-length CNF1 (D1-5) and its various regions (D1-4, D5) with BCAM. D shows the co-localization results of full-length EGFP- (D1-5) and its various regions (D1-4, D5) with BCAM-mRuby on the SKOV3 cell membrane. E shows the affinity analysis results of CNF1 D5C866S with SKOV3 cells.

[0030] Figure 2 This is a schematic diagram (A) of the CNF1 D5-based T cell conjugate constructed in Example 2, and an SDS-PAGE analysis result (B) of the purified T cell conjugate.

[0031] Figure 3 The results of the hemolysis assay (Figure B) and statistical graph (Figure A) of red blood cells after 24 hours of incubation following a specified treatment are shown. Figure B is a representative image of red blood cells after centrifugation. The color of the supernatant reflects the release of hemoglobin, while the cell precipitate represents intact red blood cells. The experimental results show that this TCE does not induce a hemolytic reaction.

[0032] Figure 4 This is a diagram showing the results of T cell binding to OV90 tumor cells and forming immune synapses mediated by T cell conjugates in Example 4; representative images are shown in Figures A and B. The white scale bar is 10 µm; C and D are statistical graphs quantifying the number of T cells and tumor cells. In the presence of TCE, more T cells attached to OV90 tumor cells and formed immune synapses. A representative image is shown in Figure B. Significant difference between TCE and the PBS control group: ****p<0.0001, ***p<0.001.

[0033] Figure 5 This is a flow cytometry analysis result of BCAM expression on solid tumor cell lines (Hep3B: human liver cancer cell line; OV90: human ovarian cancer cell line; SKOV3: human ovarian cancer cell line; NCI-N87: human gastric cancer cell line; HeLa: human cervical cancer cell line; HCT-116: human colon cancer cell line), 293T cells, human erythrocytes (RBCs) from healthy donors, and peripheral blood mononuclear cells (PBMCs) in Example 5.

[0034] Figure 6 This is a schematic diagram of the in vitro luciferase-based killing experiment of T cells mediated by T cell conjugates to kill tumor cells in Example 5.

[0035] Figure 7 This is a diagram showing the results of an in vitro luciferase-based killing experiment of T cells mediated by T cell conjugates to kill tumor cells Hep3B and OV90 in Example 5.

[0036] Figure 8 This is a diagram showing the results of an in vitro luciferase-based killing experiment of T cells mediated by T cell conjugates to kill various tumor cells and 293T cells in Example 5.

[0037] Figure 9 This is a graph showing the evaluation results of the antitumor activity of T cell conjugates in a mouse xenograft model reconstructed from activated human T cells in Example 6. A is a schematic diagram of the OV90 xenograft model treated with T cells, TCE, or T cells + TCE. B is a graph showing bioluminescence imaging results indicating that T + TCE treatment significantly reduced tumor size in mice. C is a graph showing the statistical results of bioluminescence intensity of tumors in each mouse. Significant difference between T + TCE and other control groups at the same concentration: **** p < 0.0001, no statistically significant difference in ns. D is a graph showing the standardized body weight of mice. No significant differences were observed between the T cell group, the TCE group, and the T cell + TCE group. E is a graph showing the survival rate of mice in each group. Detailed Implementation

[0038] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are only preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used is conventional equipment, and the equipment materials used in each embodiment are the same. In the following embodiments, unless otherwise specified, % refers to volume percentage.

[0039] Example 1: Analysis of the interaction between Escherichia coli toxin CNF1 D5 and BCAM

[0040] Structural analysis of the CNF1 D5-BCAM complex

[0041] Figure 1 A is a schematic diagram of the CNF1 domains, which contain multiple domains. Full-length CNF1 D5 and the extracellular domain (V1-C1) of BCAM were expressed and purified separately. These two proteins were mixed at a 1:1 molar ratio and then subjected to crystal screening and optimization. X-ray diffraction data were collected and analyzed, finally yielding the 2.9 Å structure of the CNF1 D5-BCAM complex, as shown below. Figure 1 As shown in B, CNF1 mainly interacts with BCAM V2 through β26 of the D5 domain and β17 of the BCAM V2 domain.

[0042] Isothermal titration calorimetry (ITC)

[0043] First, full-length CNF1 (SEQ ID NO.5), CNF1 D5, CNF1 D1-4 (SEQ ID NO.6), and BCAM extracellular domain (V1-C3) protein (SEQ ID NO.7) were expressed and purified. In this study, the specific steps of the ITC experiment are as follows: (1) Full-length CNF1, different CNF1 truncated variants (CNF1 D5, CNF1 D1-4), and BCAM protein samples were placed into mini dialysis cups and dialyzed overnight in ITC buffer (20 mM HEPES pH 7.5, 100 mM NaCl) to ensure that the buffer system for all proteins was completely consistent. (2) Before the reaction process began, the sample cell and injection needle were rinsed with ITC buffer, the protein samples were diluted with ITC buffer, and centrifuged at 4°C and 12000 rpm for 5 min. (3) BCAM was placed in the sample cell, with a loading volume of approximately 270 μL. Place a full-length CNF1 or a different CNF1 truncated body in the injection needle, with a sample volume of approximately 70 μL. (4) Insert the injection needle containing the full-length CNF1 or a different CNF1 truncated body into the sample cell containing BCAM, and set the titration parameters: reaction temperature 25℃; stirring speed 750 rpm; 19 titrations in total; volume of each drop 2 μL (0.4 μL for the first drop); titration duration 4.0 s (0.8 s for the first drop); interval between two titrations 150 s (60 s between the first and second drops). (5) After titration, perform fitting analysis on the titration data to obtain the fitting curve, Kd value, and other information. The results are as follows: Figure 1As shown in Figure C, CNF1 D1-5 and CNF1 D5 have essentially the same affinity for BCAM, while CNF1 D1-4 does not bind to BCAM.

[0044] Colocalization analysis of CNF1 and BCAM on SKOV3 cell membrane

[0045] SKOV3 cells overexpressing BCAM-mRuby in the logarithmic growth phase and in good condition were digested with trypsin, and the trypsin was discarded. The cells were then resuspended in complete culture medium at 2 × 10⁻⁶. 4 Cells were seeded at a density of [number] cells per cell culture medium on cell crawling slides and incubated overnight at 37°C with 5% CO2. After cell attachment, 5 µM of full-length EGFP-CNF1 or different truncated variants were added, and the cells were incubated at 4°C for 30 min. After incubation, excess unbound protein was washed away with pre-cooled PBS. 300 µL of 4% paraformaldehyde was added to each well for fixation at room temperature for 15 min, followed by two washes with PBS. The slides were mounted with DAPI-containing mounting medium. Images were observed and acquired using a Nikon laser confocal scanning microscope. Results are as follows: Figure 1 As shown in Figure D, EGFP-CNF1 D1-5, CNF1 D5, and BCAM-mRuby co-localize on the SKOV3 cell membrane. CNF1 D1-4, however, cannot co-localize with BCAM-mRuby on the SKOV3 cell membrane, further indicating that the E. coli toxin CNF1 binds to BCAM via CNF1 D5.

[0046] Affinity analysis of CNF1 D5 and SKOV3 cells

[0047] Different concentrations of EGFP-CNF1 D5 purified in vitro were compared with 3.0 × 10⁻⁶ D⁻¹. 5 SKOV3 cells were incubated at 4°C for 30 min. After incubation, excess unbound protein was washed away with PBS, and finally, the fluorescence signal of EGFP-CNF1 D5 bound to the cell surface was detected using a CytoFlex flow cytometer. Results are as follows: Figure 1 As shown in E, CNF1 D5 has an affinity of approximately 4.4 nM for SKOV3 cells (10 times stronger than that of general antibodies), which is consistent with the ITC titration results.

[0048] In summary, CNF1 D5 is a fragment that binds to BCAM with specificity and high affinity.

[0049] This invention discards fragments such as CNF1 D1-4 that have other functions and epitopes, and uses CNF1 D5, which binds to BCAM only with specificity and high affinity, as the antigen-binding domain for binding to BCAM. This can achieve highly efficient and specific targeting of BCAM without introducing potential problems caused by other functions or epitopes on CNF1, and has high safety. It provides a novel molecular and cellular strategy for immunotherapy based on a high-affinity non-antibody scaffold for malignant tumors with high BCAM expression.

[0050] Example 2: Construction and preparation of bispecific T cell conjugates

[0051] 1.1 Construction of Bispecific T Cell Conjugator Plasmid

[0052] like Figure 2 As shown, a bispecific T-cell conjugate capable of efficiently guiding T cells to kill tumor cells overexpressing BCAM was constructed by linking the anti-CD3 single-domain antibody scFv sequence (SEQ ID NO.4) to the C886S mutant sequence of the CNF1 D5 domain of *E. coli* (SEQ ID NO.2, which eliminates enzyme activity to further ensure safety). The CD3 scFv-CNF1 D5 C866S fragment was ligated to a modified pCDNA3.1 vector (containing an Igκ signal peptide at the N-terminus and a 6xHis tag at the C-terminus) via homologous recombination. After transformation, single clones were selected, plasmids were extracted, and sequencing yielded protein expression plasmids with the correct sequence.

[0053] 1.2 Expression and purification of bispecific T cell conjugate protein

[0054] The day before transfection, administer 2.0 × 10 6 293F cells were seeded at a density of 1 / mL, and the cell density reached 4.0 × 10⁶ cells / mL on the second day of culture. 6Transfection was performed at a concentration of / mL. Prepare the Opti-MEM / DNA mixture (based on a 1 L cell system) in a 50 mL centrifuge tube: 20 mL Opti-MEM + 1000 μg of the plasmid with the correct sequence obtained in step 1.1; and the Opti-MEM / PEI mixture: 20 mL Opti-MEM + 3000 μg PEI. After mixing, incubate at room temperature for 5 min. Slowly add the Opti-MEM / PEI mixture to the Opti-MEM / DNA mixture, incubate at room temperature for 15 min, and then add the mixture to the 293F cells to be transfected. 24 h after transfection, add 5 mM sodium butyrate and continue culturing for 48 h in a 37℃, 120 rpm incubator containing 8% CO2. After 48 h, collect the cell supernatant. Pre-equilibrate Ni-NTA beads with lysis buffer containing 20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 15 mM imidazole, and 0.1 mM PMSF. The supernatant was incubated with Ni-NTA beads at 4°C for 1.5 h. After washing away excess unbound proteins with lysis buffer, the bispecific T-cell conjugate protein was eluted from the Ni-NTA beads with a buffer containing 20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 500 mM imidazole, and 0.1 mM PMSF. The protein was further purified using a Superdex 200 Increase 10 / 300 column and gel filtration buffer containing 20 mM Tris-HCl pH 8.0 and 200 mM NaCl. The obtained protein was then analyzed by SDS-PAGE. Figure 2 As shown in B, TCE appears at the theoretical size (61 kDa) and has high purity.

[0055] Example 3: Safety evaluation of T-cell conjugates

[0056] Hemolysis test:

[0057] To assess the hemolytic risk associated with TCE molecules, an in vitro hemolysis assay was performed using freshly isolated human peripheral blood erythrocytes. Human peripheral blood was collected, and erythrocytes were obtained by centrifugation and washed three times with cold PBS until the supernatant was clear and colorless. The erythrocytes were resuspended in RPMI-1640 complete medium containing 10% heat-inactivated fetal bovine serum to prepare a 2% (v / v) packed erythrocyte suspension. 500 μL of this suspension was then added to: 0.1% Triton X-100 (positive control), culture medium (negative control), and 2 × 10⁻⁶ ppm. 5T cells were incubated with 0, 1.25, and 2.5 nM scFv CD3-D5 C866S protein at 37°C and 5% CO2 for 24 hours. After incubation, the cells were centrifuged, and 50 μL of the supernatant was carefully transferred from each well to a 96-well clear plate. The absorbance at 540 nm (OD540) was measured using a microplate reader to reflect the amount of hemoglobin released. The percentage of hemolysis was calculated using the following formula: Hemolysis rate (%) = (OD540 sample − OD540 negative control) / (OD540 positive control − OD540 negative control) × 100%. The experiment was independently repeated three times. The results are as follows: Figure 3 As shown in A and 3B, the color of the erythrocyte supernatant in the TCE-treated group was similar to that of the negative control, and the hemolysis rate was not significantly increased, indicating that CD3 scFv-CNF1 D5 C866S TCE does not cause hemolysis of erythrocytes within the effective killing concentration range and has good erythrocyte compatibility.

[0058] Example 4: Evaluation of the ability of T cell conjugates to bridge T cells with BCAM-positive tumor cells

[0059] TCE mediates the binding of T cells to tumor cells and the formation of immune synapses:

[0060] The ability of TCE to bridge T cells with BCAM-positive tumor cells and the formation of immune synapses were observed using confocal fluorescence microscopy.

[0061] 5×10 4 One OV90 cell was seeded on a coverslip and cultured overnight to allow it to adhere. The next day, 2 × 10⁶ cells were added. 5 Individual T cells were added to either 1 nM scFvCD3-D5 C866S TCE or an equal volume of PBS (control) and co-cultured for 2 hours. The supernatant was carefully aspirated to remove unbound suspension T cells.

[0062] Cells adhering to coverslips were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 / PBS for 10 minutes, and blocked with 3% BSA / 0.25% Triton X-100 / PBS at room temperature for 1 hour. Primary and secondary antibody incubation was performed sequentially: CD3 (Proteintech) labeled T cells, Granzyme B (BioLegend) labeled cytotoxic effector molecules, phalloidin-Alexa Fluor 488 (Beyotime) stained the actin backbone, and 647-conjugated goat anti-rabbit IgG (ThermoFisher) was used for detection. Finally, the slides were mounted with DAPI-containing mounting medium (abcam).

[0063] Images were observed and acquired using a Nikon laser confocal scanning microscope, and the number of adherent T cells and their binding events with tumor cells were quantified using ImageJ software.

[0064] Result: As Figure 4 As shown, compared with the PBS control group, significantly more T cells attached to and aggregated on the surface of OV90 tumor cells in the presence of TCE. Figure 4 A, 4B), the number of T cell-tumor cell binding events and the number of adhering T cells were significantly increased ( Figure 4 C, 4D, ****p<0.0001). Simultaneously, the polarized distribution of Granzyme B was observed at the contact site between T cells and tumor cells, indicating the successful formation of a functional immune synapse. This confirms that TCE can efficiently and specifically promote the formation of immune synaptic structures between T cells and BCAM-positive target cells, providing a structural basis for subsequent specific killing.

[0065] Example 5: In vitro functional evaluation of T cell conjugates

[0066] Analysis of BCAM expression on the surface of tumor cell lines and normal blood cells:

[0067] BCAM surface expression in the human solid tumor cell lines and normal blood cells used in Example 5 was identified by flow cytometry. All flow cytometry experiments were performed according to standard sample processing and staining procedures, and data were acquired using CytoFLEX and CytoFLEXS flow cytometers (Beckman Coulter). Specific staining was performed using a PE-labeled anti-human CD239 (BCAM) antibody (BioLegend), and background signal levels were determined using a PE-IgG2b isotype control (BioLegend). For analysis, single populations of viable cells were selected by gating using forward scatter (FSC) and side scatter (SSC) characteristics. Expression levels were expressed as Δmedian fluorescence intensity (ΔMFI = specific staining MFI − isotype control MFI).

[0068] The cells examined included: solid tumor cell lines Hep3B (human liver cancer), OV90 (human ovarian cancer), SKOV3 (human ovarian cancer), NCI-N87 (human gastric cancer), HeLa (human cervical cancer), and HCT-116 (human colon cancer), as well as 293T cells, human erythrocytes (RBCs) from healthy donors, and peripheral blood mononuclear cells (PBMCs). Results are as follows... Figure 5As shown, solid tumor cell lines Hep3B (human liver cancer), OV90 (human ovarian cancer), SKOV3 (human ovarian cancer), NCI-N87 (human gastric cancer), HeLa (human cervical cancer), and HCT-116 (human colon cancer) all exhibit high BCAM expression on their surfaces. In contrast, the major blood cell components RBC and PBMC show extremely low expression levels. The negative results for RBC and PBMC, in direct contrast to the high expression in tumor cell lines, demonstrate that the TCE of this invention possesses specificity for targeting tumors and good blood compatibility. This provides direct evidence for demonstrating the therapeutic window and reducing off-target safety risks.

[0069] Chemiluminescence assay for in vitro lethality:

[0070] This embodiment uses chemiluminescence immunoassay to detect the in vitro killing activity of the CD3 scFv-CNF1 D5 C866S bispecific T cell conjugate (TCE) against BCAM-overexpressing tumor cells. The experiment is as follows: Figure 6 As shown, it specifically includes:

[0071] Target cell preparation

[0072] Tumor cell lines stably expressing firefly luciferase (Hep3B: human hepatocellular carcinoma cell line; OV90: human ovarian carcinoma cell line; SKOV3: human ovarian carcinoma cell line; NCI-N87: human gastric carcinoma cell line; HeLa: human cervical carcinoma cell line; HCT-116: human colon cancer cell line) and the 293T control cell line were selected. Target cells were cultured in DMEM complete medium containing 10% fetal bovine serum at 37°C and 5% CO2 until the logarithmic growth phase, then digested, counted, and adjusted to the required density.

[0073] Effector cell preparation

[0074] Human peripheral blood-derived T cells were activated and expanded with CD3 / CD28 antibodies and used as effector cells for killing experiments. Effector cells and target cells were co-cultured at an effector-to-target ratio of 4:1 (E:T=4:1), with a fixed number of target cells of 5000 cells per well and the number of effector cells replenished accordingly.

[0075] Experimental grouping and treatment

[0076] CD3 scFv-CNF1 D5 C866S TCE was added to the co-culture system at different concentrations. A control group without TCE (TCE=0 group) and a negative control group were set up, which replaced TCE with an equimolar concentration of CNF1 D5 C866S monomer (unfused anti-CD3 scFv), and were also co-cultured with target cells at an effector-to-target ratio of 4:1.

[0077] Detection steps

[0078] All treatment groups were incubated together at 37℃ in a 5% CO2 cell culture incubator for 18 h. After incubation, 100 μL of D-luciferin potassium salt substrate (D-luciferin, 1.5 mg / mL) was added to each well, and the reaction was carried out in the dark for 3-5 min. The chemiluminescence signal (relative light units, RLU) of each well was detected using a multi-mode microplate reader. The number of viable cells was directly proportional to the chemiluminescence intensity, thus reflecting the amount of residual target cells.

[0079] Data processing

[0080] Lethality is calculated using the following formula:

[0081] Kill efficiency (%) = (1 − experimental group RLU₍experimental₎ / TCE=0 group RLU₍control₎) × 100%

[0082] In the experimental group, RLU₍experiment₎ represents the chemiluminescence value measured in wells with a specific concentration of TCE, while RLU₍control₎ in the TCE=0 group represents the chemiluminescence value measured in co-culture wells (T cells + target cells only) without TCE.

[0083] Experimental results

[0084] The results are as follows Figure 7-8 As shown, CD3 scFv-CNF1 D5 C866S TCE exhibited concentration-dependent high-efficiency killing activity against tumor cells with high BCAM expression, clearing most target cells at higher concentrations; however, it failed to effectively kill 293T cells with low BCAM expression, even at higher concentrations. In contrast, the negative control group, which only added CNF1 D5 C866S protein (without anti-CD3), showed no significant killing effect on either OV90 or Hep3B target cells. Figure 7 This indicates that the killing effect of TCE strictly depends on the dual-function recognition that simultaneously bridges CD3 and BCAM. This result confirms that TCE constructed based on the CNF1 D5 C866S domain can efficiently and specifically mediate the killing of BCAM-positive tumor cells by T cells.

[0085] Example 7: In vivo functional evaluation of T cell conjugates

[0086] This embodiment uses an NCG mouse peritoneal tumor model to evaluate the in vivo antitumor activity of CD3 scFv-CNF1 D5 C866S TCE combined with T cells against BCAM-overexpressing tumors.

[0087] 1. Establishment and grouping of animal models

[0088] Six- to eight-week-old female NCG-immunodeficient mice were selected and intraperitoneally inoculated with 500,000 stable OV90 tumor cells (OV90-luc) expressing firefly luciferase on day -5. Five days later, tumor colonization was confirmed by in vivo bioluminescence imaging (BLI imaging). The tumor-forming mice were randomly divided into three groups (n=5-6 per group):

[0089] • T group (T cell monotherapy control group);

[0090] • TCE group (TCE monotherapy group);

[0091] • T+TCE group (TCE combined with T cell experimental group).

[0092] 2. Dosing regimen

[0093] Each group according to Figure 9 The drug administration flowchart shown in A indicates intraperitoneal injection (ip), and the specific protocol is as follows:

[0094] Group T: 5×10⁵ injections were administered on days 0 and 4. 6 10 T cells; injected with an equal volume of PBS on days 2, 7, 9, and 16; injected with 2×10 T cells on days 11 and 14. 6 T cells.

[0095] • TCE group: 200 pmol TCE was injected on days 0 and 4; 200 pmol TCE was injected on days 2, 7, 9, 11, 14 and 16.

[0096] • T+TCE group: 5×10⁵ injections were administered on days 0 and 4. 6 One T cell combined with 200 pmol TCE; 200 pmol TCE was injected on days 2, 7, 9, and 16; 2 × 10 T cells were injected on days 11 and 14. 6 One T cell combined with 200 pmol TCE.

[0097] 3. Testing and Evaluation

[0098] Following drug administration, all mice underwent in vivo bioluminescence imaging (BLI) weekly to monitor tumor growth. The specific procedure was as follows: mice were anesthetized with isoflurane and intraperitoneally injected with D-luciferin potassium substrate (D-luciferin, 150 mg / kg). After 8 minutes of reaction, the mice were placed in an IVIS Spectrum in vivo imaging system for imaging, and the bioluminescence intensity (total photons, photons / s / cm²) was quantitatively analyzed. 2 / sr). Simultaneously, changes in mouse body weight were recorded in each group to assess the tolerability of the treatment regimen.

[0099] 4. Data Processing and Statistical Analysis

[0100] The bioluminescence intensity of tumors was plotted over time (days) to reflect the tumor growth trend in each group of mice. Statistical analysis of data from different groups was performed using GraphPad Prism software.

[0101] Experimental results

[0102] In vivo antitumor activity test results are as follows Figure 9 As shown.

[0103] Depend on Figure 9 In vivo imaging images of B (days 7, 13, and 20) show that after successful tumor colonization was confirmed on day 4, obvious bioluminescent signals could be detected in the peritoneal cavity of mice in all groups. Figure 9 Quantitative results of tumor growth curves in mice showed that, over time, the bioluminescence intensity of peritoneal tumors in mice treated with T cells alone (T group) or TCE alone (TCE group) showed a rapid upward trend, indicating continuous and rapid tumor growth. In contrast, the bioluminescence intensity of tumors in the TCE combined with T cell administration group (T+TCE group) remained at a very low level, significantly inhibiting tumor growth. A significant decrease in tumor signal was observed on day 20, which was statistically significantly different from the single-drug groups (****P<0.0001).

[0104] Figure 9 The mouse weight monitoring curves for D showed that the standardized body weight of each group remained stable at around 1.0 during the administration period. There were no significant differences between the groups and no significant weight loss or animal death was observed, indicating that the administration regimen had good safety and tolerability in mice.

[0105] Figure 9 Kaplan-Meier survival analysis of E mice showed that T+TCE treatment prolonged the survival time of mice, with a statistically significant difference compared with the single-drug group (**P<0.01).

[0106] In summary, the results of this embodiment demonstrate that the bispecific T cell conjugate constructed based on the CNF1 D5 C866S domain can efficiently and specifically bridge T cells to kill OV90 tumor cells in vivo, exhibiting excellent in vivo anti-tumor growth and clearance capabilities.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antigen-binding domain that binds to the cell surface receptor BCAM, characterized in that, The amino acid sequence of the antigen-binding domain of the cell surface receptor BCAM is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.

2.

2. The use of the antigen-binding domain of the cell surface receptor BCAM as described in claim 1 in the preparation of an immune product for treating cancer or autoimmune diseases, characterized in that, The immune product is an immune cell conjugate; wherein, the end of the immune cell conjugate that recognizes tumor-associated antigens is the antigen-binding domain of the cell surface receptor BCAM as described in claim 1.

3. The application according to claim 2, characterized in that, The immune cell conjugate is a T cell conjugate, an NK cell conjugate, or a γδ T cell conjugate.

4. The application according to claim 3, characterized in that, The T-cell conjugate is a bispecific T-cell conjugate, with the other end recognizing CD3 on T cells.

5. The application according to claim 2, characterized in that, The cancers mentioned are ovarian cancer, liver cancer, lung cancer, stomach cancer, breast cancer, colorectal cancer, prostate cancer, or skin cancer.

6. A bispecific T-cell conjugate, characterized in that, The bispecific T-cell conjugate specifically recognizes cell surface receptor BCAM at one end and CD3 of T cells at the other end; wherein the end that specifically recognizes cell surface receptor BCAM is the antigen-binding domain of cell surface receptor BCAM as described in claim 1.

7. The bispecific T cell conjugate according to claim 6, characterized in that, The amino acid sequence that identifies the CD3 domain is shown in SEQ ID NO.

3.

8. A polynucleotide, characterized in that, The polynucleotide encodes the bispecific T-cell conjugate as described in claim 6; the polynucleotide comprises a first nucleic acid and a second nucleic acid; the first nucleic acid has the sequence as described in SEQ ID NO. 4 or a variant thereof; the second nucleic acid has the sequence as described in SEQ ID NO. 2 or a variant thereof.

9. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide of claim 8.

10. A transformant, characterized in that, The transformant expresses the bispecific T cell conjugate as described in any one of claims 6-7, or contains the polynucleotide as described in claim 8, or contains the recombinant expression vector as described in claim 9.

Images