An antibody or its antigen-binding fragment that targets and recognizes mCD155 and its applications

Abstract

This invention belongs to the field of antigen recognition technology, specifically relating to an antibody or its antigen-binding fragment that targets and recognizes mCD155, and its applications. The antibody targeting and recognizing mCD155 includes sequences as shown in SEQ ID NO:8 and SEQ ID NO:16 or as shown in SEQ ID NO:24 and SEQ ID NO:32; it has a strong binding affinity to the mCD155-his protein; and CAR-T cells constructed based on this antibody exhibit excellent killing (apoptotic) activity against mCD155-mediated tumors.

Description

Technical Field

[0001] This invention belongs to the field of antigen recognition technology, specifically relating to an antibody or its antigen-binding fragment that targets and recognizes mCD155 and its applications. Background Technology

[0002] CD155 (also known as PVR, NECTIN-2, or Necl-5) is a multifunctional single-pass transmembrane protein that plays a crucial role in various biological processes. It plays a dual role in immune regulation. On the one hand, as a ligand for DNAM-1, CD155 can activate NK cells and T cells, promoting the killing effect of immune cells; on the other hand, by binding to immune checkpoint receptors such as TIGIT and CD96, CD155 can inhibit the activation and function of immune cells, forming an immunosuppressive tumor microenvironment.

[0003] Existing research indicates that CD155 expression is typically significantly upregulated in various tumor types, becoming a common characteristic of many tumor categories. High CD155 expression regulates tumor progression by influencing processes such as cell proliferation, migration, and adhesion. Specifically, it can promote cell proliferation by activating specific signaling pathways (such as the Ras-Raf-MEK-ERK signaling pathway); simultaneously, it can interact with growth factor receptors, further affecting cell proliferation. Regarding cell migration, CD155 can participate in multiple tumor-associated signaling pathways, inducing epithelial-mesenchymal transition in tumor cells and promoting tumor cell migration. Furthermore, CD155 inhibits the activation and function of NK cells and T cells by binding to immune checkpoint receptors such as TIGIT and CD96, reducing the killing ability of immune cells against tumor cells. In addition, it can promote the recruitment and activation of immunosuppressive cells (such as MDSCs and Tregs), further exacerbating tumor immune escape.

[0004] Given the crucial role of CD155 in tumor immune escape, it has become one of the important targets for tumor immunotherapy. Currently, developing antibodies specifically targeting CD155 to block its binding to immune checkpoint receptors and restore the activity and function of immune cells is one of the immunotherapy strategies targeting CD155 and its receptors. Summary of the Invention

[0005] This invention designs an antibody that can target and recognize mouse CD155 antigen (i.e., mCD155), which can specifically bind to mCD155 and has a strong binding ability.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0007] This invention provides an antibody that targets and recognizes mCD155, comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region includes light chain CDR1, light chain CDR2, and light chain CDR3. It selects any one of the following antibodies:

[0008] (a) First antibody: The amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO:1, the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID NO:3; the amino acid sequence of light chain CDR1 is shown in SEQ ID NO:9, the amino acid sequence of light chain CDR2 is shown in SEQ ID NO:10, and the amino acid sequence of light chain CDR3 is shown in SEQ ID NO:11;

[0009] (b) Second antibody: The amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO:17, the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO:18, and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID NO:19; the amino acid sequence of light chain CDR1 is shown in SEQ ID NO:25, the amino acid sequence of light chain CDR2 is shown in SEQ ID NO:26, and the amino acid sequence of light chain CDR3 is shown in SEQ ID NO:27.

[0010] Preferably, among the above-mentioned antibodies that target and recognize mCD155,

[0011] In the first antibody, the heavy chain variable region further includes heavy chain FR1 with the sequence shown in SEQ ID NO:4 and / or heavy chain FR2 with the sequence shown in SEQ ID NO:5 and / or heavy chain FR3 with the sequence shown in SEQ ID NO:6 and / or heavy chain FR4 with the sequence shown in SEQ ID NO:7; and / or the light chain variable region further includes light chain FR1 with the sequence shown in SEQ ID NO:12 and / or light chain FR2 with the sequence shown in SEQ ID NO:13 and / or light chain FR3 with the sequence shown in SEQ ID NO:14 and / or light chain FR4 with the sequence shown in SEQ ID NO:15; or

[0012] In the second antibody, the heavy chain variable region further includes heavy chain FR1 with the sequence shown in SEQ ID NO:20 and / or heavy chain FR2 with the sequence shown in SEQ ID NO:21 and / or heavy chain FR3 with the sequence shown in SEQ ID NO:22 and / or heavy chain FR4 with the sequence shown in SEQ ID NO:23; and / or the light chain variable region further includes light chain FR1 with the sequence shown in SEQ ID NO:28 and / or light chain FR2 with the sequence shown in SEQ ID NO:29 and / or light chain FR3 with the sequence shown in SEQ ID NO:30 and / or light chain FR4 with the sequence shown in SEQ ID NO:31.

[0013] Preferably, among the above-mentioned antibodies that target and recognize mCD155,

[0014] In the first antibody, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16; or

[0015] In the second antibody, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:32.

[0016] Another aspect of the present invention provides an antigen-binding fragment comprising the antibody that targets and recognizes mCD155 as described in the present invention.

[0017] In another aspect, the present invention provides a CAR structure targeting mCD155, comprising an antibody that targets and recognizes mCD155 as described in the present invention or an antigen-binding fragment as described in the present invention.

[0018] In another aspect, the present invention provides a nucleotide sequence encoding an antibody that targets and recognizes mCD155, an antigen-binding fragment, or a CAR structure.

[0019] In another aspect, the present invention provides an expression vector comprising the nucleotide sequence described herein.

[0020] In another aspect, the present invention provides an engineered cell comprising the expression vector described herein.

[0021] In another aspect, the present invention provides the use of an antibody that targets and recognizes mCD155, an antigen-binding fragment, a CAR structure, an expression vector, or an engineered cell in the preparation of a medicament for treating mCD155-mediated cancers.

[0022] In another aspect, the present invention provides the use of an antibody that targets and recognizes mCD155, an antigen-binding fragment, an expression vector, or an engineered cell in the present invention in the preparation of products for qualitative and / or quantitative detection of mCD155.

[0023] The beneficial effects of this invention include at least the following:

[0024] (1) The EC50 values ​​of the first antibody and the second antibody in the antibody that targets and recognizes mCD155 provided by the present invention are 7.156 nM and 2.737 nM respectively, indicating that they have a strong binding ability to mCD155-his protein.

[0025] (2) The CAR-T cells constructed from the antibody that targets and recognizes mCD155 provided by this invention have excellent killing (apoptotic) effects on mCD155-mediated tumors, and the proportion of apoptotic cells can reach 40%, which is significantly higher than that of the blank control group (the proportion of apoptotic cells is about 7%). Attached Figure Description

[0026] Figure 1 To detect the serum titer of anti-human CD155 antibody in New Zealand rabbits using ELISA;

[0027] Figure 2 For flow cytometry separation of IgG + mCD155 + 7AAD - Cell condition;

[0028] Figure 3 Antibody screening results for the Berkeley Lights' Beacon system;

[0029] Figure 4 The gel electrophoresis results of the amplification products;

[0030] Figure 5 The binding of different concentrations of 7041G4-scFv and 10165G6-scFv with mCD155-his is shown.

[0031] Figure 6 The binding of different concentrations of 10165G6-scFv to cell lines overexpressing CD155;

[0032] Figure 7 The constructed CAR structure plasmid map;

[0033] Figure 8 The APC fluorescence signal can be used to detect the CAR positivity rate and CAR expression intensity;

[0034] Figure 9To detect cell apoptosis at different effect-to-target ratios. Detailed Implementation

[0035] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0036] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0037] In this invention, the term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and an antibody analogue, which typically includes at least a portion of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs; the fragment of the antibody retains at least some of the binding specificity of the parent antibody.

[0038] This invention provides an antibody that targets and recognizes mCD155, comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region includes light chain CDR1, light chain CDR2, and light chain CDR3. It selects any one of the following antibodies:

[0039] (a) First antibody: The amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO:1, the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID NO:3; the amino acid sequence of light chain CDR1 is shown in SEQ ID NO:9, the amino acid sequence of light chain CDR2 is shown in SEQ ID NO:10, and the amino acid sequence of light chain CDR3 is shown in SEQ ID NO:11;

[0040] (b) Second antibody: The amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO:17, the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO:18, and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID NO:19; the amino acid sequence of light chain CDR1 is shown in SEQ ID NO:25, the amino acid sequence of light chain CDR2 is shown in SEQ ID NO:26, and the amino acid sequence of light chain CDR3 is shown in SEQ ID NO:27.

[0041] It should be noted that the antibody targeting mCD155 in this invention can be either the first antibody or the second antibody. In addition, CDR3 of the heavy chain and light chain of the above-mentioned antibody belongs to the hypervariable region of the variable region. The hypervariable region of the heavy chain and light chain of the antibody also contains two hypervariable regions, CDR1 and CDR2. The hypervariable regions of CDR1 and CDR2 are relatively stable compared with CDR3.

[0042] In some specific examples, in the first antibody targeting mCD155 described above, the heavy chain variable region further includes heavy chain FR1 with the sequence shown in SEQ ID NO:4 and / or heavy chain FR2 with the sequence shown in SEQ ID NO:5 and / or heavy chain FR3 with the sequence shown in SEQ ID NO:6 and / or heavy chain FR4 with the sequence shown in SEQ ID NO:7; and / or the light chain variable region further includes light chain FR1 with the sequence shown in SEQ ID NO:12 and / or light chain FR2 with the sequence shown in SEQ ID NO:13 and / or light chain FR3 with the sequence shown in SEQ ID NO:14 and / or light chain FR4 with the sequence shown in SEQ ID NO:15; or

[0043] In some specific examples, the second antibody targeting mCD155 described above further includes, in the heavy chain variable region, heavy chain FR1 as shown in SEQ ID NO:20 and / or heavy chain FR2 as shown in SEQ ID NO:21 and / or heavy chain FR3 as shown in SEQ ID NO:22 and / or heavy chain FR4 as shown in SEQ ID NO:23; and / or light chain variable region further includes light chain FR1 as shown in SEQ ID NO:28 and / or light chain FR2 as shown in SEQ ID NO:29 and / or light chain FR3 as shown in SEQ ID NO:30 and / or light chain FR4 as shown in SEQ ID NO:31.

[0044] It should be noted that the FR regions (FR1, FR2, FR3, FR4) in the aforementioned antibodies are backbone regions used to connect to the CDR regions and are relatively stable. In addition to the sequences mentioned above, the FR regions of the first antibody can also be selected from sequences with ≥80% identity to the FR1, FR2, FR3, and FR4 sequences, such as ≥80%, 85%, 90%, or 95%. These sequences can be derived from mouse or zoo animals (e.g., rabbits, pigs, etc.). Furthermore, the sequences of the FR regions of the heavy and light chains can be arranged in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 to constitute the heavy chain variable regions and light chain variable regions of the two antibodies mentioned above, respectively.

[0045] It should also be noted that, in addition to the aforementioned heavy chain variable region and light chain variable region, the antibody in this invention also includes a heavy chain constant region in the heavy chain and a light chain constant region in the light chain. The heavy chain constant region and light chain constant region can be derived from mouse or other animal sources (such as rabbit or pig sources), and are regions that are almost impossible to mutate. Furthermore, the heavy and light chains of the antibody may also include a signal peptide, which can facilitate antibody transmembrane penetration; the signal peptide can be a signal peptide known in the art.

[0046] In some specific examples, in the antibodies that target and recognize mCD155, the amino acid sequence of the heavy chain variable region in the first antibody is as shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:16; or in the second antibody, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:24, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:32.

[0047] It should be noted that the heavy chain variable region and light chain variable region of the first antibody and the second antibody in this invention preferably include the above-mentioned CDR region and the preferred FR region; that is, the amino acid sequence of the heavy chain variable region in the first antibody can be as shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region can be as shown in SEQ ID NO:16; the amino acid sequence of the heavy chain variable region in the second antibody can be as shown in SEQ ID NO:24, and the amino acid sequence of the light chain variable region can be as shown in SEQ ID NO:32.

[0048] This invention also provides an antigen-binding fragment comprising the antibody that targets and recognizes mCD155 as described in this invention.

[0049] In another aspect, the present invention provides a CAR structure targeting mCD155, comprising an antibody that targets and recognizes mCD155 as described in the present invention or an antigen-binding fragment as described in the present invention.

[0050] It should be noted that the antibody targeting mCD155 or the antigen-binding fragment of this invention can be used as the extracellular target antigen-binding domain of the CAR structure to prepare the CAR structure. Modifying T cells with the CAR structure can achieve precise killing of tumor cells. Furthermore, other regions of the CAR structure are well-known in the art; for example, the CAR structure also includes a hinge region, a transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain can be one or more; the amino acid sequences of each region can be sequences known in the art.

[0051] This invention also provides a nucleotide sequence that encodes an antibody targeting mCD155, an antigen-binding fragment, or a CAR structure.

[0052] It should be noted that the nucleotide sequence in this invention can be obtained by translating the above-mentioned antibody targeting mCD155, the antigen-binding fragment in this invention, or the CAR structure in this invention using conventional methods; alternatively, it can also be a nucleotide sequence after translation of the above-mentioned amino acid sequence and subsequent modification; the modification method is a method known in the art to increase expression efficiency or other nucleotide modification methods for specific purposes.

[0053] This invention also provides an expression vector comprising the nucleotide sequence described in this invention.

[0054] It should be noted that the expression vector used in this invention is selected from any one of lentiviral expression vectors, retroviral expression vectors, adenoviral expression vectors, adeno-associated virus expression vectors, DNA vectors, RNA vectors, and plasmids. Lentiviral vectors may be selected from the following groups: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedivirus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0055] This invention also provides an engineered cell that includes the expression vector described in this invention.

[0056] It should be noted that the engineered cells can be host cells, and the above expression vector is introduced into the host cells for encoding to obtain the antibody of the present invention.

[0057] The present invention also provides the use of an antibody that targets and recognizes mCD155, an antigen-binding fragment, a CAR structure, an expression vector, or an engineered cell in the preparation of a drug for treating mCD155-mediated cancer.

[0058] It should be noted that, in the above applications, mCD155-mediated cancers can be all mCD155-mediated cancers known in the art, including but not limited to B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myeloid leukemia.

[0059] This invention also provides the application of an antibody targeting mCD155, an antigen-binding fragment, an expression vector, or engineered cells in the preparation of products for qualitative and / or quantitative detection of mCD155.

[0060] It should be noted that the antibody that targets and recognizes mCD155 in this invention can target and recognize mCD155. Therefore, by adding auxiliary detection reagents to this antibody, a product for detecting mCD155 expression can be prepared. The product can be a reagent, a kit, or a microfluidic chip. The auxiliary detection reagents are reagents known in the art, such as buffer solutions, coating solutions, or indicators.

[0061] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0062] I. Immunization of mouse CD155 antigen

[0063] This embodiment is used to obtain a rabbit-derived monoclonal antibody against mouse CD155, wherein the purified recombinant CD155 extracellular region his fusion protein (mCD155-his, whose amino acid sequence (SEQ ID NO:33) is as follows):

[0064] MAQLARATRSPLSWLLLLFCYALRKAGGDIRVLVPYNSTGVLGGSTTLHCSLTSNENVTITQ

[0065] ITWMKKDSGGSHALVAVFHPKKGPNIKEPERVKFLAAQQDLRNASLAISNLSVEDEGIYECQ

[0066] IATFPRGRSRSTNAWLKVQARPKNTAEALEPSPTLILQDVAKCISANGHPPGRISWPSNVNGSH

[0067] REMKEPGSQPGTTTVTSYLSMVPSRQADGKNITCTVEHESLQELDQLLVTLSQPYPPENVSI

[0068] Using the purified antigen (SGYDGNWYVGLTNLTLTCEAHSKPAPDMAGYNWSTNTGDFPNSVKRQGNMLLISTVEDGLNNTVIVCEVTNALGSGQGQVHIIVKEKPENMQQNTRHHHHHH) as the antigen, New Zealand rabbits (12 weeks old, purchased from Chongqing Tengxin, weighing approximately 2 kg) were immunized. The rabbits were immunized three times using the purified antigen and complete Freund's adjuvant. Serum titers were detected using ELISA, following the steps outlined in patent CN117186215A, to obtain New Zealand rabbits with the highest serum titers of anti-mouse CD155 immunoglobulin. Results are as follows: Figure 1 As shown, the potency of both New Zealand rabbits, #1 and #2, is greater than 1 million.

[0069] II. Flow Cytometry Separation and Activation of Rabbit Antigen Memory B Cells

[0070] (1) Take 5 ml of peripheral blood from New Zealand rabbits with the highest serum titer of anti-human CD155 antibody, add lymphocyte separation medium (manufacturer: Cedarlane, catalog number: CL5115), and follow the instructions for lymphocyte separation medium to obtain PBMCs, 1.65*10 7 Cells, viability 99%;

[0071] (2) The PBMCs prepared above were subjected to flow cytometry staining using surface markers, as detailed below: The PBMCs were resuspended in 1 ml of FACS buffer (1.5% FBS, 2 mM EDTA, PBS buffer), and counted using a cell counter. The experiments were performed according to the materials and recommended dosages in Table 1 below; specifically, mCD155-his-biotin protein was incubated at 4°C for 1 h, followed by washing twice with FACS buffer. The remaining antibodies were added according to the dosages described in Table 1, and the mixture was incubated at 4°C for 30 min, followed by washing twice. IgG was then separated by flow cytometry. + mCD155 + 7AAD - Cells, results as follows Figure 2 As shown;

[0072] (3) IgG separated by flow cytometry + mCD155 + 7AAD - Cells were resuspended in activation medium (stemcell, 100-0645) and activated according to the manufacturer's instructions.

[0073] Table 1. Raw materials used

[0074] mark company Item number Volume (per 1*10^6 cells) mCD155-his-biotin Righteousness and righteousness in China 50259-M41H-B 4ug IgDFITC Biolegend 348206 2ul 7-AADViabilityStainingSolution Invitrogen 00-6993-50 2ul SAv-APC Biolegend 405226 2ul

[0075] III. Screening of Antibody-Secreting Cells

[0076] After 5 days of activation culture in activation medium, activated mCD155-specific memory B cells were screened using the Berkeley Lights' Beacon system. Specifically, this included harvesting cells and automatically introducing them into an OptoSelect 11k chip according to the system instructions. OEP technology was used to transfer B cells to individual nano-liter chambers (nanopenings). Using this light-based operation, all activated B cells were introduced one by one into different wells for functional screening: In the first round of screening, 6μm-8μm mCD155-conjugated microbeads (052-00053, BLI) were introduced into the chip channels, and anti-rabbit IgG fluorescent secondary antibody (Jacksonimmuno, 111-546-144) was added to identify mCD155 antigen-positive cells. In the second round of screening, rabbit IgG capture microbeads (Spherotech, RPFc-60-5) were introduced, and anti-rabbit IgG fluorescent secondary antibody (Jacksonimmuno, 111-546-144) was added to identify cells capable of secreting antibodies. The results are as follows: Figure 3 As shown, a single activated antibody-secreting cell is introduced into each well. The well is topped with antigen-coated microbeads, and the liquid is fluorescent secondary antibody. When a certain area brightens like a flower blooming, it indicates that the cell in that well can secrete an antibody that specifically recognizes the antigen. This "flowering" phenomenon becomes more pronounced over time. A total of 10491 wells on the chip contain cells, of which 406 wells secrete antibodies, and 176 wells secrete antibodies that specifically bind to mouse CD155 antigen. A total of 96 cells were exported to a 96-well plate for single-cell PCR.

[0077] IV. Amplification and recovery of VH / VL using single-cell PCR technology

[0078] Positive B cells screened using the single-cell optical fiber system were exported to a 96-well plate, yielding a total of 96 cells. Single-cell PCR was used to amplify and recover the intracellular antibody heavy and light chains. The specific procedures were as follows: First, 10 μL of RNAClean XP beads kit was added to each well to purify and separate RNA according to the manufacturer's instructions; then, the RNA was amplified and recovered using Opto... TM cDNA was synthesized and amplified using the Plasma B Discovery cDNA Synthesis Kit; the cDNA was further amplified to VH / VL using the Opto B DiscoverySanger Prep Kit; the final amplification products were analyzed by gel electrophoresis and sent to Kexin sequencing. Some gel electrophoresis results are shown below. Figure 4As shown, the 100bp DNA molecular weight standard (MD109) was used as a marker, with the heavy chain around 720bp and the light chain around 650bp.

[0079] V. Expression and purification of scFv

[0080] The above sequencing sequences were analyzed (https: / / www.imgt.org / ), yielding a total of 72 sequences. Two randomly selected antibody sequences, 7041G4 and 10165G6, were sent to Qingke Biosynthesis for expression verification and then constructed (transfected) into the PCDNA3.4 vector. Subsequently, stab-forming bacteria were obtained, and plasmid extraction was performed according to general molecular biology methods described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* (original version). Specifically, this included:

[0081] (1) One day before transfection (Day-1), the cell density of ExpiCHO-S cells was adjusted to (3-4)×10⁻⁶. 6 / ml, incubate overnight at 37℃, 8% CO2, shaking at 120 rpm; on day 0, cells grew to 7 × 10⁶ cells / ml. 6 / mL-1×10 7 / mL, dilute cells to 6×10⁶ cells / mL using fresh, preheated ExpiCHO medium. 6 / mL;

[0082] (2) Take 40 μg of the above endotoxin-free antibody-related plasmid (PCDNA3.4-B03) and add it to 2 mL of 4℃ pre-cooled OptiPRO medium. Mix well to obtain a diluted antibody plasmid solution. Take 160 μL of transfection reagent (manufacturer: Mirusbio, catalog number: MIR6270) and add it to 1.86 mL of 4℃ OptiPRO medium. Mix well to obtain a diluted transfection reagent.

[0083] (3) Add the diluted transfection reagent to the diluted antibody plasmid solution, mix well, and incubate at room temperature for 15 min to obtain DNA-liposome complex.

[0084] (4) Add the DNA-liposome complex to 30 mL of ExpiCHO-S cells, mix well, and then add to 150 mL of cell shake flask. Incubate at 37°C, 8% CO2, and shake at 120 rpm for 18-22 h to obtain transfected cells.

[0085] (5) Mix 300 μL of ExpiFectamine CHO Enhancer (transfection enhancement reagent) and 8 mL of ExpiCHO Feed (ExpiCHO) TMAfter mixing the expression medium, immediately add the transfected cells as described above, mix well, and culture at 32°C, 5% CO2, and 120 rpm for 8 days. Collect different cell culture supernatants.

[0086] (6) Filter the cell culture supernatant once with a 0.22um filter, load the protein A affinity chromatography column into the protein purifier, wash with 5 column volumes of water, then equilibrate with 8 column volumes of PBS buffer, let the sample flow through the affinity chromatography column, and then equilibrate with PBS buffer until the UV value no longer changes. Collect the eluted sample by adding elution buffer at a flow rate of 1 ml / min; concentrate the eluted antibody sample solution by ultrafiltration and change the buffer to obtain purified antibodies 7041G4-scFv and 10165G6-scFv; add 5% mannitol and 5% trehalose to the purified antibodies, aliquot them, and store them at -20℃ for long-term storage.

[0087] V. Antibody-antigen protein binding analysis

[0088] (I) Analysis of the binding ability of antibody-antigen proteins

[0089] For the antibody-antigen protein binding analysis, purified scFv antibodies (7041G4-scFv and 10165G6-scFv) were used in an ELISA experiment with mCD155-hi recombinant protein. The specific experimental steps are as follows:

[0090] (1) Dilute mCD155-his with PBS to 1ug / mL, add 100uL to each well of the ELISA plate for coating, and incubate overnight at 4℃.

[0091] (2) Discard the supernatant coating solution and wash with PBST;

[0092] (3) Block with PBST containing 1% BSA at 37°C for 2 hours;

[0093] (4) Discard the sealing solution in the well and wash with PBST;

[0094] (5) Add different concentrations of 7041G4-scFv-biotin and 10165G6-scFv-biotin antibodies diluted 1:3 with 1% BSA solution, and incubate at 37°C for 1 h.

[0095] (6) Discard the liquid and wash with PBST;

[0096] (7) Add diluted HRP secondary antibody (Beyotime, P0012A) to each well and incubate at 37°C for 1 hour;

[0097] (8) Discard the liquid and wash with PBST; add TMB chromogenic solution to each well, react at room temperature for 10-15 min, add stop solution to terminate the reaction, read the OD value at 450 nm, plot the graph and analyze it with GraphPad Prism 9, calculate the EC50 value (the concentration of the antibody when the OD450 value is half of the highest value is the EC50 value of the antibody. The smaller the EC50 value, the stronger the affinity of the antibody and the higher the binding ability to mCD155-his protein).

[0098] The results are as follows Figure 5 As shown, the binding of 7041G4-scFv and 10165G6-scFv to mCD155-his was concentration-dependent, with EC50 values ​​of 7.156 nM and 2.737 nM, respectively.

[0099] (II) Analysis of the antibody's ability to bind to cell lines overexpressing CD155

[0100] The ability of antibodies to bind to cell lines overexpressing CD155 was analyzed using flow cytometry. The specific steps are as follows:

[0101] (1) Virus packaging and cell line construction: HEK293T cells were packaged at a ratio of 5 × 10⁻⁶ cells / cells. 5 10 cells / well were seeded into a 6-well plate and cultured overnight in DMEM medium without antibiotics;

[0102] (2) Discard the culture medium before transfection and add 1 mL of fresh DMEM culture medium without antibiotics;

[0103] (3) PCDH-CMV-mouseCD155-EF1-RFP-T2A-Puro (mouseCD155 was constructed into the vector PCDH-CMV-MCS-EF1-RFP-T2A-Puro provided by Qingke Biotechnology and sent to Qingke Biotechnology for synthesis; CD155 sequence adopted uniprotaccession:Q91WP1, 1M-408R), pMD2G vector and psPAX2 vector (3ug in total) were added to 200uL of serum-free DMEM medium at a mass ratio of 2:1:1.

[0104] (4) After mixing, let stand for 16 minutes, and then add all the liquid to the six-well plate containing HEK293T cells.

[0105] (5) After culturing for 6 hours, discard the culture medium and add fresh complete DMEM culture medium for further culturing;

[0106] (6) 48 h after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter (Millipore) to obtain the virus supernatant;

[0107] (7) Add all the obtained viral supernatants to a solution containing 5×10 5 Add 4 μg / mL of polybrene (Sigma) to a 6-well plate containing HEK293T cells and incubate for 12 h.

[0108] (8) The supernatant was then discarded and fresh complete DMEM medium was added; the resulting cells were HEK293T-hCD155.

[0109] (9) Dilute the HEK293T-mCD155 cells to 2×10⁻⁶ with PBS. 6 Add 10165G6-scFv antibody at a volume of 100 μL / tube to a 1.5 mL EP tube, add serially diluted 10165G6-scFv antibody, and incubate at 4 °C for 60 min.

[0110] (10) Add 1 mL of PBS to the EP tube, centrifuge at 4℃ and 3500 rpm for 5 min, discard the supernatant, and wash the precipitate with PBS again.

[0111] (11) After centrifugation, discard the supernatant, resuspend the cells in 100 μL / tube of PBS, add 0.1 μL / tube of HisTagAntibody[iFluor 647] (GenScript, A01802), and incubate at 4°C in the dark for 30 min.

[0112] (12) Wash twice with PBS, centrifuge and discard the supernatant;

[0113] (13) Resuspend the cells in 200 μL / tube of PBS, detect them by flow cytometry, and analyze them by plotting with GraphPad Prism 9;

[0114] The results are as follows Figure 6 As shown, the results indicate that antibody 10165G6-scFv can bind to cell lines overexpressing CD155 in a concentration-dependent manner, with an EC50 value of 3.96 nM; antibody 7041G4-scFv can bind to cell lines overexpressing CD155 in a concentration-dependent manner, with an EC50 value of 11.42 nM.

[0115] VI. CAR Structure Construction and Testing

[0116] (I) Lentiviral preparation

[0117] The 10165G6-scFv antibody sequence was sent to a vector manufacturer for CAR structure plasmid construction. The plasmid map is shown below. Figure 7As shown, the original component is EF1A>CD8-leader / 1016G6 / CD8-hinge / CD8-TM / 4-1BB-CD3zeta-SFFV>mCherry. The stab bacteria were obtained from the company, and plasmids were extracted by streaking and shaking according to common molecular biology methods. The sequence of the prepared CAR structure is shown in Table 2 below.

[0118] Table 2 Sequences of CAR structures

[0119] name Base sequence Kozak SEQ ID NO:34 CD8-Leader SEQ ID NO:35 10165G6 SEQ ID NO:36 CD8-hinge SEQ ID NO:37 CD8-TM SEQ ID NO:38 4-1BB SEQ ID NO:39 CD3zeta SEQ ID NO:40

[0120] In addition, viral supernatant was obtained by viral packaging method in the analysis of antibody binding ability to cell lines overexpressing CD155 as described in section 5(2) above.

[0121] (II) CAR Positive Rate Test

[0122] Mouse spleens and some lymph nodes were harvested, ground, and lymphocytes were separated using gradient centrifugation. After centrifugation, mononuclear cell layers were washed with physiological saline and cultured in RPMI 1640 complete medium containing 10% FBS to obtain mouse lymphocytes. The obtained lymphocytes were activated for 24 hours with anti-CD3.CD28 monoclonal antibody, and the virus supernatant prepared in the above example was added to the activated cells for infection. On the third day of viral infection, the CAR-T positivity rate was detected by flow cytometry. The detected protein was biotinylated mouse CD155 protein, and the APC fluorescent secondary antibody for streptavidin (Biolegend, 405207) was also detected. The ScFv sequence of the CAR antigen recognition region can recognize biotinylated mouse CD155 protein, and biotin can bind to the streptavidin APC fluorescent secondary antibody. Therefore, the APC fluorescence signal can be used to detect the CAR positivity rate and CAR expression intensity. The results are as follows: Figure 8 As shown, the results indicated that the CAR positivity rate was 70% three days after viral transduction.

[0123] VII. Detection of the in vitro killing ability of CAR-T cells against different tumor cells

[0124] The prepared CAR-T cells and B16 cells were centrifuged at 500g for 3 minutes, then resuspended in 1640 medium (5% FBS) and counted. Then, CAR-T cells and B16 melanoma cells were mixed in the same well according to the effector-to-target ratio (CAR-T cell to B16 cell ratio) E:T = 1:1, 1:2, and 1:4, with one replicate per well. The control group consisted of the same number of activated T cells mixed with the same number of target cells in the same well. All cells were incubated at 37°C for 4 hours and analyzed using eBioscience. TMAnnexin V apoptosis detection kit (Invitrogen, 88-8102-72) was used, and the detection was performed according to the kit instructions. The percentage of Annexin V positive results was compared. The results are as follows: Figure 9 As shown, the results indicate that there were significant statistical differences between the two groups when the effective-to-target ratios E:T were 1:1, 1:2, and 1:4.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that targets and recognizes mCD155, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, the light chain variable region comprising a light chain CDR1, a light chain CDR2, and a light chain CDR3; characterized in that, selects any one of the following antibodies or antigen-binding fragments thereof: (a) the first antibody or antigen-binding fragment thereof: the amino acid sequence of heavy chain CDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of heavy chain CDR2 is as shown in SEQ ID NO: 2, and the amino acid sequence of heavy chain CDR3 is as shown in SEQ ID NO: 3; the amino acid sequence of light chain CDR1 is as shown in SEQ ID NO: 9, the amino acid sequence of light chain CDR2 is GAF, and the amino acid sequence of light chain CDR3 is as shown in SEQ ID NO: 11; (b) the second antibody or antigen-binding fragment thereof: the amino acid sequence of heavy chain CDR1 is as shown in SEQ ID NO: 17, the amino acid sequence of heavy chain CDR2 is as shown in SEQ ID NO: 18, and the amino acid sequence of heavy chain CDR3 is as shown in SEQ ID NO: 19; the amino acid sequence of light chain CDR1 is as shown in SEQ ID NO: 25, the amino acid sequence of light chain CDR2 is AVS, and the amino acid sequence of light chain CDR3 is as shown in SEQ ID NO:

27.

2. The antibody or antigen-binding fragment thereof targeting mCD155 according to claim 1, wherein, in the first antibody or antigen-binding fragment thereof, the heavy chain variable region further comprises a heavy chain FR1 having a sequence as shown in SEQ ID NO: 4 and / or a heavy chain FR2 having a sequence as shown in SEQ ID NO: 5 and / or a heavy chain FR3 having a sequence as shown in SEQ ID NO: 6 and / or a heavy chain FR4 having a sequence as shown in SEQ ID NO: 7; and / or the light chain variable region further comprises a light chain FR1 having a sequence as shown in SEQ ID NO: 12 and / or a light chain FR2 having a sequence as shown in SEQ ID NO: 13 and / or a light chain FR3 having a sequence as shown in SEQ ID NO: 14 and / or a light chain FR4 having a sequence as shown in SEQ ID NO: 15; or in the second antibody or antigen-binding fragment thereof, the heavy chain variable region further comprises a heavy chain FR1 having a sequence as shown in SEQ ID NO: 20 and / or a heavy chain FR2 having a sequence as shown in SEQ ID NO: 21 and / or a heavy chain FR3 having a sequence as shown in SEQ ID NO: 22 and / or a heavy chain FR4 having a sequence as shown in SEQ ID NO: 23; and / or the light chain variable region further comprises a light chain FR1 having a sequence as shown in SEQ ID NO: 28 and / or a light chain FR2 having a sequence as shown in SEQ ID NO: 29 and / or a light chain FR3 having a sequence as shown in SEQ ID NO: 30 and / or a light chain FR4 having a sequence as shown in SEQ ID NO:

31.

3. The antibody or antigen-binding fragment thereof targeting mCD155 according to claim 2, wherein, in the first antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 16; or in the second antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

32. ​ ​ ​ The amino acid sequence of the heavy chain variable region in the second antibody or antigen binding fragment thereof is shown as SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:

32.

4. A CAR targeting mCD155, characterized in that, The second antibody or antigen binding fragment thereof in the antibody or antigen binding fragment thereof for targeted recognition of mCD155 according to any one of claims 1 to 3.

5. A nucleic acid molecule, characterized in that, The antibody or antigen binding fragment thereof for targeted recognition of mCD155 according to any one of claims 1 to 3.

6. A nucleic acid molecule, characterized in that, The CAR according to claim 4.

7. An expression vector comprising the nucleic acid of claim 1. The nucleic acid molecule according to claim 5.

8. An expression vector, characterized by, The nucleic acid molecule according to claim 6.

9. An engineered cell, comprising, The expression vector according to claim 7.

10. An engineered cell, comprising: The expression vector according to claim 8, wherein the engineered cell is a T cell.

11. Use of the CAR according to claim 4 or the nucleic acid molecule according to claim 6 or the expression vector according to claim 8 or the engineered cell according to claim 10 in the preparation of a medicament for treating melanoma.

12. Use of the antibody or antigen binding fragment thereof for targeted recognition of mCD155 according to any one of claims 1 to 3 in the preparation of a product for qualitative and / or quantitative detection of mCD155.

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