Bispecific single-chain antibody PC5-VB8 and application of bispecific single-chain antibody PC5-VB8 in preparation of medicine for treating prostatic cancer

Abstract

The invention belongs to the field of preparation of genetically engineered drugs and antibody drugs, and relates to a bispecific single-chain antibody PC5-VB8 and application thereof in preparation of drugs for treating prostatic cancer. The bispecific single-chain antibody disclosed by the invention is shown as SEQ ID NO. 8. The bispecific single-chain antibody can specifically bind to tumor cells expressing PSCA, neutralize VEGF and block growth of endothelial cells and formation of endothelial tubes. In a prostatic cancer mouse model, the effect of inhibiting tumor growth is enhanced by combined treatment of the prostatic cancer vaccine and an engineered phage vaccine. The bispecific single-chain antibody not only can reduce the generation of tumor blood vessels, but also can significantly remodel the phenotype-function spectrum of tumor infiltration immune cells in combination with the engineered bacteriophage vaccine, NK cells and M1 macrophages are increased, MDSC cells are reduced, and the expression profiles of surface functional molecules of cDC, CD4 + and CD8 + T cell subsets significantly shift towards the anti-tumor direction.

Description

Technical Field

[0001] This invention belongs to the field of gene-engineered drugs and antibody drug manufacturing, and relates to the bispecific single-chain antibody PC5-VB8 and its application in the preparation of prostate cancer treatment drugs. Background Technology

[0002] The tumor microenvironment (TME), composed of immune cells, stromal cells, and the extracellular matrix, is a crucial site for the interaction between the tumor and the host immune system during tumorigenesis. The immunosuppressive effects of the TME severely hinder the effective anti-tumor immune response of therapeutic cancer vaccines. The TME can directly inhibit the immunological function of dendritic cells (DCs), preventing the body from effectively initiating a specific immune response. This is a significant factor contributing to the failure of DC-based cancer vaccines to achieve the expected immunogenicity. Numerous cytokines present in the TME, including VEGF, interleukin-10 (IL-10), and transforming growth factor-β (TGF-β), can affect the normal immunological function of DCs. Among these cytokines, VEGF plays a particularly prominent role in tumorigenesis and development. Firstly, it not only promotes tumor angiogenesis and increases vascular permeability but also binds to VEGF receptors on the cell surface, activating downstream signaling pathways such as receptor tyrosine kinases (RTKs) and neuropilins (NRPs) to promote tumor development. More importantly, VEGF can significantly inhibit the differentiation, maturation, and motility of DCs, and affect CD8. + T cell immune function plays a crucial role in inducing immune tolerance in the body. Studies have shown that inhibiting VEGF or its downstream signaling pathways can eliminate its immunosuppressive function, promote the differentiation and maturation of dendritic cells (DCs), and enhance the body's anti-tumor immune response. Therefore, VEGF plays a significant role in tumor development and the body's anti-tumor immune response.

[0003] Currently, the combination of anti-VEGF antibodies (such as bevacizumab) and immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies) has shown synergistic efficacy in multiple tumor types. However, the systemic administration of these antibodies, which neutralizes the main side effects caused by VEGF in the circulatory system and tissues, hinders the wider application of this approach. Local or targeted administration can significantly reduce blood drug concentrations, thereby greatly reducing systemic toxicity. Prostate stem cell antigen (PSCA), a tumor-associated antigen discovered in 1998, has high prostate tissue specificity. It is expressed at low levels in normal prostate tissue but at high levels in prostate cancer tissue, and is associated with androgen independence and the formation of metastatic cancer. Furthermore, PSCA protein molecules are located on the cell surface, making them less susceptible to inducing immune tolerance, thus making them an ideal target for the immunodiagnosis and treatment of prostate cancer.

[0004] Therefore, this invention utilizes antibody library technology and phage display technology to screen single-chain specific antibodies against PSCA and VEGF, constructing therapeutic bispecific antibodies. Leveraging their dual-function properties, these antibodies target tumors that accumulate in large quantities at the tumor site while simultaneously interfering with the immunosuppressive tumor microenvironment, aiming to break the tumor's immune escape mechanism and restore and enhance the anti-tumor immune response within the tumor microenvironment. This is combined with engineered phage vaccines to explore a novel synergistic tumor immunotherapy modality. Summary of the Invention

[0005] The purpose of this invention is to provide a bispecific single-chain antibody PC5-VB8, the sequence of which is shown in SEQ ID NO. 8.

[0006] Another object of the present invention is to provide a complex comprising a bispecific single-chain antibody PC5-VB8 and an engineered phage, wherein the sequence of the bispecific antibody is shown in SEQ ID NO. 8, and the engineered phage is a phage displaying the antigenic epitope shown in SEQ ID NO. 15.

[0007] The final object of the present invention is to provide the use of the bispecific single-chain antibody PC5-VB8 or the above-described complex in the preparation of a prostate cancer therapeutic agent.

[0008] To achieve the above objectives, the present invention employs the following technical measures:

[0009] Obtaining the bispecific single-chain antibody PC5-VB8:

[0010] The applicant constructed recombinant expression vectors for PSCA and VEGF, respectively, to express PSCA (shown in SEQ ID NO.1) and VEGF165 proteins (shown in SEQ ID NO.2), and used these as antigens to immunize Balb / c mice. Hybridoma cells were prepared using hybridoma technology, resulting in 14 anti-PSCA monoclonal hybridoma cell lines and 6 anti-VEGF monoclonal hybridoma cell lines. Antibody library technology was used to construct libraries with a size of 4.16 × 10⁻⁶ cells. 5 PFU, 3.2 × 10 5 PFU anti-PSCA and anti-VEGF single-chain antibody gene libraries were used. After three rounds of phage display screening, the anti-PSCA and anti-VEGF single-chain antibody libraries were specifically enriched by 55-fold and 10.4-fold, respectively. Clones were randomly selected from the third screening library and identified by scFv ELISA and Sanger sequencing analysis to determine the anti-PSCA single-chain antibody PC5 and the anti-VEGF single-chain antibody VB8, which had the highest abundance and the highest affinity.

[0011] The sequence of the single-chain antibody PC5 is shown in SEQ ID NO.3, and the gene encoding it is shown in SEQ ID NO.4. Its heavy chain variable region is shown in SEQ ID NO.9, and its light chain variable region is shown in SEQ ID NO.10.

[0012] The sequence of the single-chain antibody VB8 is shown in SEQ ID NO.5, and the gene encoding it is shown in SEQ ID NO.6. Its heavy chain variable region is shown in SEQ ID NO.11, and its light chain variable region is shown in SEQ ID NO.12.

[0013] The scope of protection of this invention includes:

[0014] The bispecific single-chain antibody PC5-VB8, the sequence of which is shown in SEQ ID NO.8.

[0015] Fusion protein obtained by fusing bispecific single-chain antibody PC5-VB8 with a protein tag.

[0016] Genes encoding the aforementioned bispecific single-chain antibodies or fusion proteins.

[0017] The gene described above, preferably, is the gene encoded by SEQ ID NO.7.

[0018] Expression cassettes, recombinant vectors, recombinant microorganisms, or in vitro recombinant cells containing the above-mentioned coding genes.

[0019] A complex containing the bispecific single-chain antibody PC5-VB8.

[0020] Preferably, the complex described above comprises engineered bacteriophages.

[0021] Preferably, the engineered bacteriophage described above is a bacteriophage displaying the antigenic epitope shown in SEQ ID NO. 15.

[0022] The use of bispecific single-chain antibody PC5-VB8, fusion protein, gene encoding bispecific single-chain antibody PC5-VB8 or fusion protein, expression cassette having the above-mentioned encoding gene, recombinant vector, recombinant microorganism or in vitro recombinant cell or complex containing bispecific single-chain antibody PC5-VB8 in the preparation of prostate cancer therapeutic drugs.

[0023] The above-described application and the drug achieve their effect by inhibiting the proliferation of prostate cancer tumor cells.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1. One of the beneficial effects of this invention is that it utilizes phage antibody library technology to construct anti-PSCA and anti-VEGF single-chain antibody gene libraries, respectively, and obtains a high-affinity anti-PSCA and anti-VEGF single-chain antibody and its encoding genes PC5 and VB8 by screening with phage display technology.

[0026] 2. The second beneficial effect of the present invention is that by linking two single-chain antibodies into a bispecific single-chain antibody, prokaryotic expression can be achieved, which is simple to operate and has low production cost.

[0027] 3. The third beneficial effect of the present invention is that the bispecific single-chain antibody has the activity of binding antigens PSCA and VEGF at the same time, which can target and bind PSCA-positive cells, and can also neutralize VEGF to inhibit endothelial cell growth and endothelial tube formation.

[0028] 4. The fourth beneficial effect of the present invention is that it provides a treatment scheme of bispecific single-chain antibody combined with engineered phage vaccine. The bispecific single-chain antibody remodels the immune microenvironment, enhances the anti-tumor immunity induced by engineered phage vaccine, and more effectively inhibits the growth of subcutaneously transplanted prostate tumors in mice. Attached Figure Description

[0029] Figure 1 To identify a panning library of anti-PSCA single-chain antibodies for scFv ELISA;

[0030] Among them: ★ marks the 20 single clones that were sequenced.

[0031] Figure 2 The binding affinity of different anti-PSCA single-chain antibodies to recombinant PSCA was detected by ELISA.

[0032] Figure 3 Western blotting was used to detect the binding ability of different anti-PSCA single-chain antibodies to PSCA in prostate cancer cells.

[0033] Figure 4 To identify a panning library of anti-VEGF single-chain antibodies for scFv ELISA;

[0034] Among them: ★ marks the 17 single clones that were sequenced.

[0035] Figure 5 The binding affinity of different anti-VEGF single-chain antibodies to recombinant VEGF was detected by ELISA.

[0036] Figure 6 For the detection of purified PC5-VB8 bispecific protein by SDS-PAGE and Western Blot;

[0037] Where A is the SDS-PAGE image of PC5-VB8 and B is the Western Blot detection diagram of PC5-VB8.

[0038] Figure 7 To analyze the binding affinity of the PC5-VB8 bispecific antibody to the antigens PSCA and VEGF for SPR analysis;

[0039] Where: A represents the binding force between PC5-VB8 and antigen PSCA, and B represents the binding force between PC5-VB8 and antigen VEGF.

[0040] Figure 8 A schematic diagram of the fluorescent labeling of PC5-VB8 bispecific antibody with PSCA-positive DU145 cells;

[0041] DU145 is a human prostate cancer cell line, and RWPE-1 is a human prostate epithelial cell line.

[0042] Figure 9 This diagram illustrates how the PC5-VB8 bispecific antibody binds to VEGF to inhibit its promotion of endothelial cell growth.

[0043] Figure 10 A schematic diagram illustrating how PC5-VB8 bispecific antibody binds to VEGF to inhibit its effect on endothelial tube formation in endothelial cells;

[0044] Among them: A: Fluorescence microscopic observation of endothelial tube formation; B: Statistical analysis of endothelial tube branching.

[0045] Figure 11 A schematic diagram of the experimental design and results of the treatment of tumor-bearing organisms with PC5-VB8 bispecific antibody;

[0046] Where: A represents the establishment of the tumor-bearing model and treatment plan; B represents the tumor growth curve of each mouse during treatment; CD represents the statistics of tumor dissection at the end of treatment; E represents the mouse weight during treatment; F represents the blood routine results; and G represents the immunohistochemical detection results of tumor tissue microvessels.

[0047] Figure 12 A schematic diagram illustrating the classification of major subsets of tumor-infiltrating immune cells;

[0048] Wherein: A is a heatmap of the expression of 42 markers in all immune cell populations, and B is the type of tumor-infiltrating immune cells that differed between the combination therapy group and the control group.

[0049] Figure 13 For fine differentiation of subgroups of MDSCs infiltrating tumors;

[0050] Where: A is the cluster heatmap; B is the MDSC subpopulation that differed from the control group in the combined treatment group; C is the maker of differences between C01 subpopulation cell groups.

[0051] Figure 14 For fine differentiation of subpopulations of tumor-infiltrating DCs;

[0052] Where: A is the cluster heatmap; B is the DC subpopulation that differed from the control group in the combined treatment group; C is the intergroup difference maker for CO3 and C11 subpopulation cells; D is the intergroup difference maker for C09 subpopulation cells.

[0053] Figure 15 For the fine differentiation of T cell subpopulations infiltrating tumors;

[0054] Where: A is the cluster heatmap; B is the T cell subpopulation that differed from the control group in the combined treatment group; C is the intergroup difference marker for the C05 subpopulation; and D is the difference marker for the C12 cell subgroup. Detailed Implementation

[0055] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0056] Example 1:

[0057] Anti-PSCA single-chain antibodies and anti-VEGF single-chain antibodies were obtained using phage display technology:

[0058] First, recombinant expression vectors for PSCA and VEGF were constructed to express PSCA (SEQ ID NO.1) and VEGF165 protein (SEQ ID NO.2), respectively. These vectors were then used as antigens to immunize Balb / c mice. Hybridoma cells were prepared using hybridoma technology, resulting in 14 PSCA-resistant monoclonal hybridoma cell lines and 6 VEGF-resistant monoclonal hybridoma cell lines. Antibody library technology was then used to construct libraries with a size of 4.16 × 10⁻⁶ cells. 5 PFU, 3.2 × 10 5 PFU anti-PSCA and anti-VEGF single-chain antibody gene library;

[0059] After three rounds of phage display screening, the anti-PSCA single-chain antibody library was enriched 55-fold in specificity. Clones randomly selected from the third round of screening were identified by scFv ELISA and analyzed by Sanger sequencing. Figure 1 As shown, 96 monoclonal antibodies were randomly selected from the third round of panning of the anti-PSCA single-chain antibody library for scFv ELISA identification to analyze their binding to the PSCA antigen. The results showed that the colorimetric values ​​of most clones were higher than those of the uncoated blank control, indicating that the specifically binding antibodies were effectively screened during the phage display panning process. Figure 2 As shown, the anti-PSCA single-chain antibody clones PC5, PD10, and PA4, which had the highest separation frequency and no internal stop codons, were tested for their binding ability to the recombinant PSCA antigen using ELISA. The results showed that all three anti-PSCA specific recombinant antibodies, PC5, PD10, and PA4, specifically bound to the prokaryotically expressed recombinant PSCA antigen. Figure 3 As shown, proteins were extracted from human prostate cancer cells PC-3. After BCA protein quantification, 40 μg of PC-3 cell protein was loaded onto the sample. Western blot analysis showed that four anti-PSCA specific recombinant antibodies, PC5, PD10, and PA4, specifically bound to PSCA in human prostate cancer cells PC-3. The single-chain anti-PSCA antibody PC5, with the highest abundance and affinity, was ultimately identified. The sequence of the single-chain antibody PC5 is shown in SEQ ID NO.3, and the gene encoding it is shown in SEQ ID NO.4. Its heavy chain variable region is shown in SEQ ID NO.9, and the light chain variable region is shown in SEQ ID NO.10.

[0060] After three rounds of phage display screening, the anti-VEGF single-chain antibody library was specifically enriched 10.4-fold. Clones randomly selected from the third screening library were identified by scFv ELISA and analyzed by Sanger sequencing. Figure 4As shown, 96 single clones were randomly selected from the third round of panning of the anti-VEGF single-chain antibody library for scFv ELISA identification to analyze their binding to the VEGF antigen. The results showed that the colorimetric values ​​of a small number of clones were higher than those of the uncoated blank control, indicating that the specifically binding antibodies were effectively screened during the phage display panning process. Sequencing analysis of the antibody sequences of 17 clones revealed three different types of antibodies. Figure 5 As shown, three different candidate antibodies, VB8, VC2, and VD8, were expressed. ELISA identification showed that all three could bind to the antigen VEGF. Given that VB8 had the highest isolation frequency, it was selected for the construction of the bispecific antibody. The sequence of the single-chain antibody VB8 is shown in SEQ ID NO.5, and the gene encoding it is shown in SEQ ID NO.6. Its heavy chain variable region is shown in SEQ ID NO.11, and the light chain variable region is shown in SEQ ID NO.12.

[0061] Example 2:

[0062] Obtaining the bispecific single-chain antibody PC5-VB8:

[0063] 1) SfiI and NcoI restriction enzyme sites were added to the 5' end of the PC5 gene, and BstEII restriction enzyme site was added to the 3' end. An AscI restriction enzyme site was added to the 5' end of the VB8 gene, and SalI and NotI restriction enzyme sites were added to the 3' end. The two single-chain antibodies were ligated using a flexible linker peptide to obtain the PC5-VB8 gene (as shown in SEQ ID NO. 7). The PC5-VB8 gene was synthesized by Nanjing GenScript and cloned into the pJET1.2 vector to obtain the pJET1.2-PC5-VB8 plasmid. The pJET1.2-PC5-VB8 plasmid and pET28b vector were digested with NcoI and NotI, respectively, purified by agarose gel electrophoresis, and the target gene was ligated into the vector using T4 DNA ligase to obtain the pET28b-PC5-VB8 recombinant vector. Transformed into E. coli BL21(DE3) competent cells by heat shock, the cells were plated on LB agar plates containing Kan (50 μg / ml) and incubated at 37°C for 15 h. Single clones were picked, cultured, and amplified by PCR using vector-specific primers T7 / T7-ter. Positive clones were identified based on DNA band size and sent to Shanghai Sangon Biotech for sequencing verification.

[0064] Recombinant E. coli BL21(DE3) / pET28b-PC5-VB8 with correct sequencing was subjected to IPTG-induced protein expression. The supernatant was purified and dialyzed to obtain the bispecific single-chain antibody PC5-VB8. SDS-PAGE and Western Blot analysis confirmed that this invention successfully achieved soluble prokaryotic expression of the PC5-VB8 bispecific antibody. Figure 6 Using this method, a bispecific antibody for PC5-VB8 with an HIS tag was obtained, containing the amino acid sequence shown in SEQ ID NO.8, with a yield of 1 mg / L of bacterial culture.

[0065] Example 3:

[0066] Detection of the binding affinity of bispecific single-chain antibody PC5-VB8 to antigens PSCA and VEGF:

[0067] 1) Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 instrument. Immediately before injection, 400 mM EDC and 100 mM NHS were mixed to prepare an activator. Antigen PSCA (50 μg / mL) and antigen VEGF (25 μg / mL) were injected at a flow rate of 10 μL / min for 100 s and 30 s respectively to fix them on the chip as ligands. Bispecific antibody PC5-VB8 was used as the flowing analytical sample, and the dissociation equilibrium constant of the bispecific antibody was determined.

[0068] 2) Dilute the bispecific antibody PC5-VB8 to eight concentrations (1000, 500, 250, 125, 62.5, 31.25, 15.62, and 0 μM) using run buffer (1×EP Buffer). Inject PC5-VB8 into the channel at a flow rate of 30 μL / min, with an contact time of 60 s and a dissociation time of 300 s. Both the association and dissociation processes were handled within the run buffer.

[0069] 3) The Bioevaluation software was used to analyze the association rate constant (Ka), dissociation rate constant (Kd), and equilibrium dissociation constant (KD) of the bispecific antibody.

[0070] The results are as follows Figure 7 As shown: SPR identification revealed that both bispecific antibodies could bind to the antigens PSCA and VEGF with high affinity, and their KD values ​​were 6.21 × 10⁻⁶. -8 M, 6.72×10 -8 M and Ka are 4.994 × 10⁻⁶ respectively. 4 M -1 ·s -1 1.408×10 5 M -1 ·s -1 Kd is 0.003101s -10.009468s -1 .

[0071] Example 4:

[0072] Immunofluorescence assay was used to verify the binding of the bispecific single-chain antibody PC5-VB8 to PSCA in prostate cancer cells.

[0073] 1) Take sterilized cell slides (25 mm in diameter) and place them in a six-well culture plate. Pretreat with 1 mg / mL poly-L-lysine and place at 37°C for at least 30 min.

[0074] 2) Take the collected DU145 cell suspension and mix it at a ratio of 10... 5 / The wells were placed on a glass slide and incubated at 37°C for 24 hours;

[0075] 3) After the slide adheres to the wall, add 2 mL of 4% PFA, incubate at room temperature for 30 min, and wash the slide three times with PBS;

[0076] 4) Block with 5% BSA at room temperature for 1 hour, then wash 3 times with PBS;

[0077] 5) Add the prokaryotically expressed bispecific recombinant antibody PC5-VB8 and incubate overnight at 4°C;

[0078] 6) Wash 3 times with PBS, add 200 μL of diluted anti-His mouse monoclonal antibody (1:800) to each well and treat for 1 h;

[0079] 7) Wash 5 times with PBS, add 200 μL of FITC-labeled goat anti-mouse IgG antibody (1:100) to each well, and incubate at room temperature in the dark for 1 h;

[0080] 8) Wash 5 times with PBS, add 100 μL of DAPI working solution to each well, and stain nuclei for 5 min in the dark;

[0081] 9) Wash 5 times with PBS, add one drop of anti-fluorescence quencher, place it on a glass slide, fix the four corners with nail polish, place it in a dark and ventilated place, and observe and photograph it under a fluorescence microscope after it dries.

[0082] The results are as follows Figure 8 As shown, the PC5-VB8 bispecific antibody can bind to PSCA-positive prostate cancer cells, while the labeled fluorescent signal is barely detectable in the control group (human prostate epithelial cells RWPE-1).

[0083] Example 5:

[0084] Inhibition assay of PC5-VB8 bispecific antibody in endothelial cells

[0085] 1) Revive cryopreserved HUVEC cells.

[0086] 2) When the third-generation cells are in good condition and the density reaches 80%, collect the cells and resuspend them in DMEM basal medium (no growth factors and FBS supplementation required).

[0087] 3) Press 10 4 Cells / well density were cultured to 96-well plates and cultured overnight (12 h).

[0088] 4) Different concentrations (0, 1, 2, 3, 4, 5 μg / ml) of bispecific antibody were mixed with a constant amount (50 ng / ml) of VEGF and incubated at 37°C for 2 h. Then the mixture was added to cells and the plate was incubated for 48 h.

[0089] 5) Add 10 μl of CCK8 reagent to each well (set up a set of blank wells with culture medium and CCK8) and incubate at 37°C for 2 h. Measure the absorbance at a wavelength of 450 nm.

[0090] The results are as follows Figure 9 As shown, the PC5-VB8 bispecific antibody can neutralize VEGF, thereby inhibiting VEGF's promotion of HUVEC cell growth.

[0091] Example 6:

[0092] PC5-VB8 Bispecific Antibody Inhibition of Endothelial Tube Formation Assay

[0093] 1) Thaw the low-growth-factor basement membrane matrix gel solution on ice. Transfer 50 μl of thawed matrix gel per well vertically to a pre-chilled 96-well cell culture plate. Incubate at 37°C for 30 min to allow the matrix gel solution to form a gel.

[0094] 2) Healthy HUVEC cells were diluted to 10⁻⁶ in DMEM basal medium (serum-free). 6 100 μl per well was added to the well plate coated with the matrix gel.

[0095] 3) Take 100 μl of serum-free DMEM medium (containing different concentrations of bispecific antibodies 0-40 μg / ml and constant VEGF 50 ng / ml) and pre-incubate for 2 h, then add it to the cell culture well.

[0096] 4) After incubation at 37℃ for 6 hours, examine the formation and condition of endothelial tubes under a high-power field of view using an inverted microscope.

[0097] 5) Carefully remove the culture medium and gently blot dry with a paper towel, being careful not to disturb the endothelial tube.

[0098] 6) Add 100 μL of detection buffer containing 0.1% Calcein-AM to each well and incubate at 37°C for 30 min.

[0099] 7) Gently wash with 100 μL of PBS and remove the wash residue as described in step 5. Repeat this step twice.

[0100] 8) Examine and image endothelial cells and endothelial tubes using a fluorescence microscope. Statistically analyze the number of endothelial tube grids using ImageJ.

[0101] The results are as follows Figure 10 As shown, the PC5-VB8 bispecific antibody can neutralize VEGF, thereby inhibiting the formation of endothelial cell tubes, and this effect is concentration-dependent.

[0102] Example 7:

[0103] Application of PC5-VB8 Bispecific Antibody in the Preparation of Drugs for Prostate Cancer Treatment

[0104] 1. Establishment of a mouse subcutaneous prostate tumor model

[0105] 1) Remove the frozen RM-1-PSA-PSCA cells from the liquid nitrogen tank, thaw them rapidly in a 37°C water bath, transfer them to a 15ml centrifuge tube, add 5ml of culture medium, and centrifuge at 200g for 3min.

[0106] 2) Remove the supernatant, add RPMI 1640 complete medium (1% penicillin and antibiotic, 10% FBS) to resuspend the cells and transfer them to a 10cm cell culture dish. Place the dish in a cell culture incubator for incubation.

[0107] 3) When the cells reach 80-90% confluence, digest them with trypsin for 3 minutes, stop the digestion with complete culture medium, wash once with PBS, and then passage them at a ratio of 1:3.

[0108] 4) After three passages, cells in good growth condition were digested with trypsin and washed twice with PBS, then resuspended in RPMI 1640 medium and the cell density was adjusted to 2 × 10⁶ cells / year. 7 / ml, placed on ice;

[0109] 5) Twenty male C57BL / 6J mice aged 6-8 weeks were selected and divided into four experimental groups (PBS group, BsAb group, DD-Phage group, and Combination group). On day 0, 0.1 mL of RM-1-PSA-PSCA cells (2 × 10⁻⁶ cells) were collected. 6 (each mouse) was injected subcutaneously into the right back of the mouse via subcutaneous injection.

[0110] 6) Starting on day 6 after tumor growth, the mice were treated according to their respective groups. DD-Phage involved injecting 100 μL of an engineered phage vaccine formulation (genetically engineered to fuse pVIII protein with PSA) into the right groin of each mouse. 65-73Antigenic epitopes are displayed on the surface of bacteriophages, 1×10 11 The pfu (pfu / mouse) group was treated with the following treatments: BsAb group received 100 μL of bispecific antibody PC5-VB8 (5 mg / kg) injected around the tumor in mice; Combination group received 100 μL of engineered phage vaccine preparation injected into the right groin of each mouse, and simultaneously received 100 μL of bispecific antibody PC5-VB8 (5 mg / kg) injected around the tumor in mice; PBS group received 100 μL of PBS solution.

[0111] The engineered phage vaccine formulation described above is a phage capable of displaying the antigenic epitope shown in SEQ ID NO. 15. In this embodiment, its preparation method is as follows:

[0112] First, the helper phage was modified to incorporate the antigenic epitope PSA. 65-73 It is fused with the gene encoding the pVIII protein, the main coat protein of filamentous bacteriophage, and its nucleic acid sequence is shown in SEQ ID NO.13, including Tac promoter, lac operator, and PSA. 65-73 and the pVIII protein gene. The recombinant pVIII protein (rpVIII) encoded by PSA is... 65-73 The antigenic epitope and pVIII protein are composed of an amino acid sequence shown in SEQ ID NO.14, where PSA... 65-73The amino acid sequence of the antigenic epitope is shown in SEQ ID NO.15. The rpVIII fusion gene was integrated into the genome of a filamentous phage (Hyperphage, purchased from Progen) that was deficient in the pIII protein gene. The phage vector was then modified to achieve complete pIII expression by removing the amber stop codon preceding the pIII protein gene in the classic phage vector (pHENHi). Finally, the modified helper phage and the phage vector were co-transformed into *E. coli* XL1-Blue MRF'. Recombinant hosts successfully co-transformed with phage vectors and helper phages were added to LB liquid medium (containing 100 μg / mL Amp and 50 μg / mL Kan) and incubated upside down at 37°C for 12–16 h. 1 mL of the overnight culture was added to 140 mL of 2TY medium (containing 100 μg / mL Amp and 50 μg / mL Kan) and incubated at 30°C with shaking at 200 rpm for 15 h. The overnight culture was aliquoted into 50 mL centrifuge tubes and centrifuged at 4°C and 4000 rpm for 30 min. The supernatant was transferred to a new 50 mL centrifuge tube, 1 / 5 volume of PEG / NaCl solution was added, and the mixture was thoroughly mixed and incubated on ice for 1 h. The tube was then centrifuged at 4°C and 8000 rpm for 30 min, and the supernatant was discarded. The precipitate was resuspended in 40 mL of LB liquid medium. Add 1 / 5 volume of PEG / NaCl solution to ddH₂O, mix thoroughly, and incubate at 4°C for 20 min. Centrifuge at 4°C and 4000 rpm for 30 min, discarding the supernatant. Briefly centrifuge to remove residual PEG / NaCl solution. Resuspend the precipitate in 3 mL of PBS containing 15% glycerol. Filter through a 0.22 μm filter membrane and store at -80°C for later use. Phage particle counting formula:

[0113] Virus particles / mL = [(A269-A320)×6×10 16 ] / (base count / virus particle)

[0114] 7) Record the tumor size and body weight of the mice every two days. Calculate the tumor volume using the formula: Tumor volume (mm²) 3 = 0.5 × tumor long diameter × tumor short diameter 2 .

[0115] 8) The first treatment was administered 6 days after tumor cell inoculation, and the treatment continued for 18 days for a total of 7 sessions (i.e., treatment was administered to each group on days 6, 8, 10, 12, 16, 20, and 24). Figure 11 (A) Samples were taken after treatment.

[0116] 2. Whole blood collection and dissection of tumor-bearing mice

[0117] 1) Blood was collected by enucleation and then sent to Wuhan Saiweier Biotechnology Co., Ltd. for routine blood testing.

[0118] 2) After blood collection, the mice were euthanized and the heart, liver, spleen, lung and kidney tissues of the tumor-bearing mice in each treatment group were taken for HE staining, microscopic examination and image acquisition.

[0119] 3) Remove tumor tissue from tumor-bearing mice. Take tumor tissue from 3 mice in each group and put it into tissue preservation solution. Store and transport it at low temperature (2-8℃) immediately. Ensure that it is delivered to Zhejiang Proton Health Technology Co., Ltd. within 48 hours after removal from the body. Detect immune cells by single-cell mass cytometry (CyTOF) using 42 immune cell surface markers (Table 1).

[0120] 4) Immunohistochemistry of the remaining tumor tissue.

[0121] To observe angiogenesis in tumor tissue, tumor sections were immunostained with anti-CD34 and anti-CD31 antibodies, and incubated overnight at 4°C. The primary antibody was washed off, and the secondary antibody was added and incubated at 37°C for 1 hour at room temperature. Images were then acquired under a microscope from the areas with the largest positive staining areas.

[0122] Table 1. Panels used in this mass spectrometry flow cytometry study.

[0123]

[0124] Results analysis:

[0125] like Figure 11 As shown, in a mouse model of prostate cancer, the first treatment was administered 6 days after tumor cell inoculation, followed by 7 treatments over 18 days. Figure 11 (A). Bispecific antibody (BsAb) combined with engineered phage (DD-Phage) vaccine enhanced the inhibitory effect on tumor growth. Figure 11 In the treatment with BD, the mice did not experience a significant decrease in body weight. Figure 11 After treatment (E), routine blood tests showed that the values ​​of monocyte count, lymphocyte percentage, monocyte percentage, and neutrophil percentage were all within the normal reference range. Figure 11(F). Simultaneously, it was found that in the Control group, the mean values ​​of RBC, HGB, and HCT in erythrocyte-related parameters (RBC, HGB, HCT, MCV, MCH, MCHC) were below the normal range, while the BsAb, DD-Phage, and Combination groups were closer to the normal reference range, thus indicating the preliminary safety of the treatment regimen. Furthermore, angiogenesis in tumor tissue was detected by HE staining and immunohistochemical staining of vascular endothelial cell markers (CD31 and CD34). The blood vessel density in tumors treated with anti-BsAb (BsAb group and Combination group) was significantly lower than in other control groups, indicating its significant anti-angiogenic effect. Figure 11 (G).

[0126] like Figure 12 As shown, combined treatment with the bispecific antibody PC5-VB8 and the engineered phage vaccine (DD-Phage) induced significant changes in tumor-infiltrating immune cells. Analysis of large cell subsets revealed an increase in NK cells, mast cells, CD8 effector T cells, and CD4 regulatory T cells, while MDSC cells decreased. These changes in cells within tumor-infiltrating tissue collectively determine the efficacy of anti-tumor immunotherapy.

[0127] like Figure 13 As shown, fine analysis of small cell subpopulations using functional molecules reveals ( Figure 13 In the combined treatment group, the number of cluster 1 tumor-infiltrating cells (with the M1 macrophage phenotype) was significantly increased compared to the control group. Figure 13 In addition, the combined treatment group showed increased MHC II expression in M1 macrophages compared to the control group, while CD27, CD44, and CD172a expression was decreased. Figure 13 (C). This indicates that these M1 macrophages can more efficiently present antigens from pathogens or tumor cells to T cells, promoting T cell proliferation and differentiation, and enhancing the body's immune surveillance and clearance capabilities against pathogens or tumors. Regarding changes in MDSC subpopulations, the CO3, CO4, CO5, CO6, CO7, CO8, and C10 cell populations in the DD-Phgae and combination therapy groups were significantly reduced compared to the control group. Meanwhile, the C13, C14, C15, and C16 cell populations were significantly increased compared to the control group. Figure 13 (B) Based on the surface markers of different cell subpopulations, the treatment group showed a significant reduction in MDSCs with higher expression of CD44 and CD172a, suggesting that these cells may have stronger cell migration and immunosuppressive capabilities. The treatment group showed an increase in MDSCs with lower expression of CD44 and CD172a, suggesting that these may be MDSCs with weak immunosuppressive capabilities or MDSCs that participate in inflammatory responses rather than immunosuppression.

[0128] like Figure 14 As shown, a detailed analysis of the DC subgroup ( Figure 14 In the combined treatment group (A), CO3 and C11 subsets of cells were significantly reduced, while CO6 and C09 subsets of cells were significantly increased. Figure 14 (B). Analysis of maker expression on cell subpopulation surfaces showed ( Figure 14 In the middle (C), CO3 exhibits a steady-state-immature / early mature cDC2 (CD86) pattern. + MHCII hi CD274 low and CD172 low C11 is represented as CD103. + cDC1 cells (CD103) + CD11c + and MHCII hi It also exhibits low expression of CD11b. C06 is an activated migratory cDC2 (Ki-67) variant. + CD103 low MHCII hi The expression of Ki67 on its surface suggests that this subtype of cells is in a state of rapid proliferation. C09 exhibits classic cDC1 (CD103) behavior. + CD11c + and MHCII hi Furthermore, the combined treatment group showed a significant increase in MHCII expression of C09. Figure 14 (D).

[0129] like Figure 15 As shown, a detailed analysis of small subsets of T cells ( Figure 15 In the combined treatment group (A), the CO2 cell subset was significantly reduced ( Figure 15 (B) CD4, exhibiting an effect / memory phenotype. + T cells (CD44) hi TCR-β-chain hi CD4 hi The C05 cell subset increased significantly. Figure 15 (B) CD4, exhibiting an activation / memory-prone tendency. + Regulatory T cells (CD44) + CD25 + TCR-β-chain hi CD3e + CD39 + CD4 hiAnalysis of the differences in maker expression levels among the subpopulation cells showed that the combined treatment group had decreased expression levels of CD38, CD44, CD127, and CD279, and increased expression levels of CD39 and CD196. Figure 15 (C). The C12 cell subset was significantly increased ( Figure 15 (B) exhibits highly activated effector CD8. + T cells (Ly-6C) hi TCRβ-chain hi CD279(PD-1) hi CD39 + CD86 hi Analysis of the differences in maker expression levels among the subpopulation cells showed that the expression levels of CD86 and CD69 in the combined treatment group were significantly higher than those in the control group. Figure 15 (D).

[0130] In summary, the bispecific antibody PC5-VB8, used alone or in combination with engineered phage vaccines, demonstrated tumor-suppressive effects in treating subcutaneous prostate cancer transplants, with good safety profiles. Mechanistic studies revealed that the combination therapy significantly reduced tumor angiogenesis density (decreased expression of CD31 and CD34). More importantly, CyTOF high-dimensional analysis revealed that the combination therapy profoundly remodeled the tumor immune microenvironment: on the one hand, it promoted the infiltration of anti-tumor immune cells, such as NK cells and M1 macrophages; on the other hand, it significantly reduced immunosuppressive myeloid-derived suppressor cells (MDSCs). Simultaneously, this strategy also drove the growth of cDC subsets and CD4+. + and CD8 + Changes in T cell subsets are moving towards anti-tumor effects.

Claims

1. A synthetic bispecific single-chain antibody PC5-VB8, the sequence of the bispecific antibody is shown in SEQ ID NO.

8.

2. A fusion protein of the bispecific single-chain antibody PC5-VB8 of claim 1 and a protein tag.

3. A gene encoding the bispecific single-chain antibody of claim 1 or the fusion protein of claim 2.

4. The gene of claim 3, which is shown in SEQ ID NO.

7.

5. An expression cassette, a recombinant vector, a recombinant microorganism or an ex vivo recombinant cell having the encoding gene of claim 3.

6. A complex containing the bispecific single-chain antibody PC5-VB8 of claim 1.

7. The composite of claim 6, wherein: The complex comprises an engineered bacteriophage.

8. The composite of claim 7, wherein: The engineered bacteriophage is a bacteriophage displaying an antigenic epitope shown in SEQ ID NO.

15.

9. Use of the bispecific single-chain antibody PC5-VB8 of claim 1, the fusion protein of claim 2, the gene of claim 3, the expression cassette, the recombinant vector, the recombinant microorganism or the ex vivo recombinant cell of claim 5 or the complex of claim 6 in the preparation of a medicament for treating prostate cancer.

10. The use of claim 9, wherein the medicament is achieved by inhibiting the proliferation of prostate cancer tumor cells.

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