Antibody for targeted recognition of mCD155 or antigen binding fragment thereof and application thereof

Abstract

The invention belongs to the technical field of antigen recognition, and particularly relates to an antibody for targeted recognition of mCD155 or an antigen binding fragment thereof and application thereof. The antibody for targeted recognition of mCD155 comprises a sequence as shown in SEQ ID NO: 8 and SEQ ID NO: 16 or a sequence as shown in SEQ ID NO: 24 and SEQ ID NO: 32. The binding capacity of the gene and mCD155-his protein is high; and the CAR-T cell constructed on the basis of the antibody has an excellent killing (apoptosis) effect on mCD155 mediated tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of antigen recognition, and in particular relates to an antibody or an antigen binding fragment thereof for targeting and recognizing mCD155 and an application thereof. Background Art

[0002] CD155 (also known as PVR, NECTIN-2 or Necl-5) is a multifunctional single-pass transmembrane protein that plays a key role in a variety of 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 and promote 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 studies have shown that in many types of tumors, the expression of CD155 is often significantly upregulated, becoming one of the common characteristics of many types of tumors. High expression of CD155 regulates tumor progression by affecting 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); at the same time, it can also interact with growth factor receptors to further affect the cell proliferation process; in terms of affecting cell migration, CD155 can participate in a variety of tumor-related signaling pathways, induce epithelial-mesenchymal transformation of tumor cells, and promote tumor cell migration. In addition, CD155 inhibits the activation and function of NK cells and T cells by binding to immune checkpoint receptors such as TIGIT and CD96, and reduces the ability of immune cells to kill tumor cells. In addition, it can also promote the recruitment and activation of immunosuppressive cells (such as MDSCs, Tregs, etc.), further exacerbating tumor immune escape.

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

[0005] The present 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] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides an antibody that targets and recognizes mCD155, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and the light chain variable region comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3; any one of the following antibodies is selected:

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

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

[0010] Preferably, in the above-mentioned antibody targeting and recognizing mCD155,

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

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

[0013] Preferably, in the above-mentioned antibody targeting and recognizing 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, which includes the antibody of the present invention that targets and recognizes mCD155.

[0017] In another aspect, the present invention further provides a CAR structure targeting mCD155, which comprises the antibody targeting and recognizing mCD155 of the present invention or the antigen binding fragment of the present invention.

[0018] In another aspect, the present invention provides a nucleotide sequence encoding the antibody targeting and recognizing mCD155 in the present invention, the antigen binding fragment in the present invention, or the CAR structure in the present invention.

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

[0020] In another aspect, the present invention provides an engineered cell, which comprises the expression vector of the present invention.

[0021] In another aspect, the present invention provides an antibody targeting and recognizing mCD155, or an antigen binding fragment, or a CAR structure, or an expression vector, or an engineered cell of the present invention, in the preparation of a drug for treating mCD155-mediated cancer.

[0022] In another aspect, the present invention provides a use of the antibody targeting and recognizing mCD155 of the present invention, or the antigen binding fragment of the present invention, or the expression vector of the present invention, or the engineered cell of the present invention in the preparation of a product for qualitative and / or quantitative detection of mCD155.

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

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

[0025] (2) The CAR-T cells constructed with the antibody targeting and recognizing mCD155 provided by the present invention have excellent killing (apoptosis) effect 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%). BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The titer of New Zealand rabbit serum against human CD155 antibody was tested by ELISA;

[0027] Figure 2 IgG + mCD155 + 7AAD - Cell condition;

[0028] Figure 3 Screening antibodies for the Berkeley Lights' Beacon system;

[0029] Figure 4 The gel electrophoresis of the amplified product;

[0030] Figure 5 The binding of different concentrations of 7041G4-scFv and 10165G6-scFv to mCD155-his;

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

[0032] Figure 7 This is the plasmid map of the constructed CAR structure;

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

[0034] Fig. 9Cell apoptosis was detected at different effector-target ratios. DETAILED DESCRIPTION

[0035] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0036] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0037] In the present invention, the term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which generally include at least part of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs; antibody fragments retain at least some of the binding specificity of the parent antibody.

[0038] The embodiment of the present invention provides an antibody that targets and recognizes mCD155, including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and the light chain variable region includes a light chain CDR1, a light chain CDR2, and a light chain CDR3; any one of the following antibodies is selected:

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

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

[0041] It should be noted that the antibody targeting and recognizing mCD155 in the present invention can be any of the first antibody and the second antibody; in addition, the CDR3 of the heavy chain and the light chain in the above-mentioned antibody belongs to the hypervariable region of the variable region, and the hypervariable region of the heavy chain and the light chain of the antibody also includes two hypervariable regions, CDR1 and CDR2, and the hypervariable regions of CDR1 and CDR2 are relatively stable relative to CDR3.

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

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

[0044] It should be noted that the FR (FR1, FR2, FR3, FR4) in the above-mentioned antibodies is a framework region, which is used to connect the CDR region and is relatively stable. In addition to the above-mentioned sequences, the FR region in the above-mentioned first antibody can also select sequences with a sequence identity of ≥80% with the above-mentioned FR1, FR2, FR3 and FR4, such as ≥80%, 85%, 90% or 95%, and the source can be mouse or zoo (such as rabbit, pig and other animal sources). In addition, the sequences of the FR regions of the above-mentioned heavy and light chains and the sequences of the CDR regions can be arranged according to FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, respectively constituting the heavy chain variable region and light chain variable region of the above-mentioned two antibodies.

[0045] It should also be noted that, in addition to the heavy chain variable region and light chain variable region mentioned above, the antibody of the present 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 the light chain constant region can be derived from mice or other animal sources (such as rabbit, pig, etc.), which are almost non-mutated regions. In addition, the heavy chain and light chain of the antibody can also include a signal peptide, which can help the antibody to penetrate the membrane; the signal peptide can be a signal peptide known in the art.

[0046] In some specific examples, in the above-mentioned antibodies targeting and recognizing mCD155, 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 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.

[0047] It should be noted that the heavy chain variable region and the light chain variable region of the first antibody and the second antibody in the present invention preferably include the above-mentioned CDR region and the preferred FR region, respectively; 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] The embodiment of the present invention further provides an antigen binding fragment, which includes the antibody of the present invention that targets and recognizes mCD155.

[0049] In another aspect, the present invention further provides a CAR structure targeting mCD155, which comprises the antibody targeting and recognizing mCD155 of the present invention or the antigen binding fragment of the present invention.

[0050] It should be noted that the antibody targeting and recognizing mCD155 in the present invention or the antigen binding fragment in the present invention can be used as the extracellular target antigen binding domain of the CAR structure to prepare the CAR structure, and the CAR structure transforms T cells to achieve precise killing of tumor cells. In addition, other regions of the CAR structure are well known in the art, such as the CAR structure also includes a hinge region, a transmembrane domain and an intracellular signaling domain, wherein the intracellular signaling domain can be one or more than two; the amino acid sequence of each region can select a sequence known in the art.

[0051] The embodiment of the present invention also provides a nucleotide sequence, which encodes the antibody targeting and recognizing mCD155 in the present invention, or the antigen binding fragment in the present invention, or the CAR structure in the present invention.

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

[0053] An embodiment of the present invention further provides an expression vector, which includes the nucleotide sequence of the present invention.

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

[0055] An embodiment of the present invention further provides an engineered cell, which includes the expression vector of the present invention.

[0056] It should be noted that the engineered cell may be a host cell, and the above expression vector is introduced into the host cell to encode the antibody of the present invention.

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

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

[0059] The embodiments of the present invention also provide a use of the antibody of the present invention that targets and recognizes mCD155, or the antigen-binding fragment of the present invention, or the expression vector of the present invention, or the engineered cell of the present invention in the preparation of a product for qualitative and / or quantitative detection of mCD155.

[0060] It should be noted that the antibody targeting and recognizing mCD155 in the present invention can target and recognize mCD155, so the antibody can be added with an auxiliary detection reagent to prepare a product for detecting the expression of mCD155, and the product can be a reagent, a kit or a microfluidic chip; the auxiliary detection reagent is a reagent known in the art, such as a buffer, a coating solution or an indicator.

[0061] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0062] 1. Immunization of mice with CD155 antigen

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

[0064] MAQLARATRSPLSWLLLLFCYALRKAGGDIRVLVPYNSTGVLGGSTTLHCSLTSNENVTITQ

[0065] ITWMKKDSGGSHALVAVFHPKKGPNIKEPERVKFLAAQQDLRNASLAISNLSVEDEGIYECQ

[0066] IATFPRGRSRSTNAWLKVQARPKNTAEALEPSPTLILQDVAKCISANGHPPGRISWPSNVNGSH

[0067] REMKEPGSQPGTTTVTSYLSMVPSRQADGKNITCTVEHESLQELDQLLVTLSQPYPPENVSI

[0068] SGYDGNWYVGLTNLTLTCEAHSKPAPDMAGYNWSTNTGDFPNSVKRQGNMLLISTVEDGLNNTVIVCEVTNALGSGQGQVHIIVKEKPENMQQNTRHHHHHH) was used as an antigen to immunize New Zealand rabbits (12 weeks old, purchased from Chongqing Tengxin, weighing about 2 kg). The New Zealand rabbits were immunized three times using the above-mentioned purified antigen and complete Freund's adjuvant. The serum titer was detected by using the ELISA method according to the steps of patent CN117186215A to obtain the New Zealand rabbits with the highest serum titer of anti-mouse CD155 immunoglobulin; the results are as follows Figure 1 As shown, the titers of the two New Zealand rabbits 1# and 2# were both greater than 1 million.

[0069] 2. Flow cytometry separation and activation of rabbit antigen memory B cells

[0070] (1) Take 5 ml of peripheral blood from the New Zealand rabbit with the highest serum titer of anti-human CD155 antibody, add lymphocyte separation solution (manufacturer Cedarlane, catalog number CL5115), and operate according to the instructions of the lymphocyte separation solution to obtain PBMC, 1.65*10 7 cells, with a viability of 99%;

[0071] (2) The PBMC prepared above was subjected to the flow cytometry surface marker staining method, which is described in detail as follows: PBMC were resuspended in 1 ml FACS buffer (1.5% FBS, 2 mM EDTA, PBS buffer), counted by a cell counter, and tested according to the materials and recommended dosages in Table 1 below; specifically including: mCD155-his-biotin protein, incubated at 4°C for 1 hour, then washed twice with FACS buffer, and the remaining antibodies were added according to the dosages described in Table 1, incubated at 4°C for 30 minutes, washed twice, and then IgG was separated by flow cytometry. + mCD155 + 7AAD - Cells, the results are as follows Figure 2 As shown;

[0072] (3) IgG separated by flow cytometry + mCD155 + 7AAD - The 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 Part Number Volume (per 1*10^6 cells) mCD155-his-biotin Sino Biological 50259-M41H-B 4ug IgDFITC Biolegend 348206 2ul 7-AADViabilityStainingSolution Invitrogen 00-6993-50 2ul SAv-APC Biolegend 405226 2ul

[0075] 3. Screening of Antibody-Secreting Cells

[0076] After 5 days of activation culture in activation medium, the activated mCD155-specific memory B cells were screened using the Berkeley Lights'Beacon system; specifically, the cells were harvested and automatically introduced into the OptoSelect 11k chip according to the system manual, and the B cells were transferred to a single nanoliter volume chamber (nanopening) using OEP technology; using this light-based operation, all activated B cells were introduced into different wells one by one, and then functional screening was performed: in the first round of screening analysis, 6μm-8μm mCD155-coupled microbeads (052-00053, BLI) were first introduced into the chip channel, and anti-rabbit IgG fluorescent secondary antibodies (jacksonimmuno, 111-546-144) were added to screen and determine the cells with positive mCD155 antigen binding; in the second round of screening, rabbit IgG capture microbeads (spherotech, RPFc-60-5) were first used, and anti-rabbit IgG fluorescent secondary antibodies (jacksonimmuno, 111-546-144) were added to screen and determine the cells that can secrete antibodies; the results are shown in Figure 2. Figure 3 As shown, a single activated antibody-secreting cell is introduced into each well, with antigen-coated microbeads above the well and fluorescent secondary antibodies in the liquid. When a certain area lights up like a flower, it means that the cells in the corresponding well can secrete antibodies that specifically recognize the antigen, and as time goes by, the flowering phenomenon becomes more and more obvious. There are a total of 10,491 wells on the chip containing cells, of which 406 wells secrete antibodies, and 176 wells secrete antibodies that can specifically bind to mouse CD155 antigens. A total of 96 cells are exported to a 96-well plate for single-cell PCR operations.

[0077] 4. Amplification and recovery of VH / VL using single cell-PCR technology

[0078] The positive B cells screened by the single-cell optical system were exported to a 96-well plate, with a total of 96 cells exported. The antibody heavy and light chains in the cells were amplified and recovered using single-cell PCR technology. The specific operation was as follows: first, 10ul RNAClean XP beads kit was added to each well to purify and isolate RNA according to the manufacturer's instructions; Opto TM Plasma B Discovery cDNA Synthesis Kit was used to synthesize and amplify cDNA; Opto B Discovery Sanger Prep Kit was used to further amplify VH / VL cDNA; the final amplified product was analyzed by gel electrophoresis and sent to Qingke for sequencing. Some of the gel electrophoresis results are shown in the figure below. Figure 4As shown, a 100 bp DNA molecular weight standard (MD109) was used as a marker, the heavy chain was around 720 bp, and the light chain was around 650 bp.

[0079] 5. Expression and purification of scFv

[0080] The above sequencing sequences were analyzed (https: / / www.imgt.org / ), and a total of 72 sequences were obtained. The antibody sequences 7041G4 and 10165G6 were randomly selected and sent to Qingke Biosynthesis for expression verification, and constructed (transfected) into the PCDNA3.4 vector. Subsequently, the puncture bacteria were obtained, and the plasmids were extracted by streaking and shaking according to the general molecular biology methods described in the Molecular Cloning Experiment Guide (Fourth Edition) (Original Version) (streak separation, shaking amplification and plasmid extraction); specifically including:

[0081] (1) One day before transfection (day -1), adjust the cell density of ExpiCHO-S cells to (3-4)×10 6 / ml, 37°C, 8% CO 2 , shaken at 120 rpm overnight; on day 0, cells grew to 7×10 6 / mL-1×10 7 / mL, dilute the cells to 6 × 10 6 / mL;

[0082] (2) Take 40ug of the above-mentioned endotoxin-free antibody-related plasmid (PCDNA3.4-B03) and add it to 2mL of 4°C pre-cooled OptiPRO medium and mix well to obtain a diluted antibody plasmid solution; take 160uL of transfection reagent (manufacturer: Mirusbio, catalog number: MIR6270) and add it to 1.86mL of 4°C OptiPRO medium and 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 minutes to obtain a DNA-liposome complex;

[0084] (4) Add the DNA-liposome complex to 30 mL of ExpiCHO-S cells, mix well and add to a 150 mL cell shake flask at 37°C and 8% CO 2 , shake and culture at 120 rpm, and obtain transfected cells 18h-22h after transfection;

[0085] (5) Add 300uL ExpiFectamine CHO Enhancer (transfection enhancement reagent) and 8mL ExpiCHO Feed (ExpiCHO TMExpression medium) was added immediately after mixing, and the above transfected cells were added and mixed, and incubated at 32°C, 5% CO 2 , after culturing with shaking at 120 rpm for 8 days, different cell culture supernatants were collected;

[0086] (6) The cell culture supernatant was filtered once with a 0.22 um filter, and the protein A affinity chromatography column was installed on a protein purifier. It was washed with water for 5 column volumes, and then equilibrated with PBS buffer for 8 column volumes. The sample was allowed to flow through the affinity chromatography column, and then equilibrated with PBS buffer until the UV value no longer changed. The eluted sample was collected by connecting the elution buffer at a flow rate of 1 ml / min; the eluted antibody sample solution was ultrafiltered and concentrated, and the solution was replaced to obtain purified antibodies 7041G4-scFv and 10165G6-scFv; 5% mannitol and 5% trehalose were added to the purified antibodies, divided into small portions, and stored at -20°C for long-term storage.

[0087] 5. Antibody-antigen protein binding analysis

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

[0089] For antibody-antigen protein binding analysis, purified scFv antibodies (7041G4-scFv and 10165G6-scFv) and mCD155-his recombinant protein were used for ELISA experiments. The specific experimental steps are as follows:

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

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

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

[0093] (4) Discard the blocking solution in the wells and wash with PBST;

[0094] (5) Add different concentrations of 7041G4-scFv-biotin and 10165G6-scFv-biotin antibodies diluted in a 1:3 gradient 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 (Biyuntian, P0012A) to each well and incubate at 37°C for 1 h;

[0097] (8) Discard the liquid and wash with PBST; add TMB colorimetric solution to each well and react at room temperature for 10-15 min. Add stop solution to terminate the reaction, read the OD value at 450 nm, make a graph and analyze it with GraphPad Prism 9 to calculate the EC50 value (the concentration of the antibody when the OD450 value is half of the maximum 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 with the mCD155-his protein).

[0098] The results are as follows Figure 5 As shown, 7041G4-scFv and 10165G6-scFv bound to mCD155-his in a concentration-dependent manner, and the EC50 values ​​were 7.156 nM and 2.737 nM, respectively.

[0099] (II) Analysis of antibody binding ability to cell lines overexpressing CD155

[0100] For the analysis of the ability of the antibody to bind to the cell line overexpressing CD155, flow cytometry was used for detection. The specific implementation steps are as follows:

[0101] (1) Virus packaging and cell line construction: HEK293T cells were cultured at 5×10 5 Cells / well were plated in six-well plates and cultured overnight in DMEM medium without double antibodies;

[0102] (2) Before transfection, discard the culture medium and add 1 mL of fresh DMEM medium without dual antibodies;

[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, and the CD155 sequence used uniprot accession: Q91WP1, 1M-408R), pMD2G vector and psPAX2 vector (3ug in total) were added into 200uL serum-free DMEM medium at a mass ratio of 2:1:1;

[0104] (4) After mixing, let stand for 16 min, and then add all the liquid to the above six-well plate with HEK293T cells;

[0105] (5) After 6 h of culture, the culture medium was discarded and fresh complete DMEM medium was added;

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

[0107] (7) All the obtained supernatants of different viruses were added to 5×10 5 In a 6-well plate of HEK293T cells, polybrene (Sigma) was added at a final concentration of 4 ug / mL and cultured for 12 h;

[0108] (8) Then discard the supernatant and add fresh complete DMEM medium; the resulting cells are HEK293T-hCD155;

[0109] (9) Dilute the HEK293T-mCD155 cells to 2×10 6 / mL, add 100μL / tube to a 1.5mL EP tube, add serially diluted 10165G6-scFv antibody, and incubate at 4℃ for 60min;

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

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

[0112] (12) Wash twice with PBS and discard the supernatant after centrifugation;

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

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

[0115] 6. CAR structure construction and testing

[0116] (I) Lentivirus preparation

[0117] The 10165G6-scFv antibody sequence was sent to the vector manufacturer for constructing the CAR structure plasmid. The plasmid map is shown in Figure 7As shown, the original is EF1A>CD8-leader / 1016G6 / CD8-hinge / CD8-TM / 4-1BB-CD3zeta-SFFV>mCherry. The punctured bacteria were obtained from the company, and then the plasmid was extracted by streaking and shaking according to the general molecular biology method; 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 according to the virus packaging method in the analysis of the ability of antibodies to bind to cell lines overexpressing CD155 in step 5 (ii) above.

[0121] (II) CAR positive rate test

[0122] The spleen and part of the lymph nodes of the mouse were taken, ground and separated by gradient centrifugation; after centrifugation, the mononuclear cell layer was washed with saline, and RPMI 1640 complete medium containing 10% FBS was added to culture to obtain mouse lymphocytes. After the obtained lymphocytes were activated by anti-CD3.CD28 monoclonal antibodies for 24 hours, the virus supernatant prepared in the above example was added to the activated cells for infection. On the third day of viral infection, the positive rate of CAR-T was detected by flow cytometry. The detection protein was biotinylated mouse CD155 protein and streptavidin APC fluorescent secondary antibody (biolegend, 405207). The ScFv sequence of the CAR antigen recognition region can recognize the biotinylated mouse CD155 protein, and biotin can bind to the streptavidin APC fluorescent secondary antibody. Therefore, the APC fluorescent signal can be used to detect the CAR positive rate and CAR expression intensity; the results are as follows Figure 8 As shown, the results showed that CAR positivity was 70% 3 days after viral transduction.

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

[0124] The prepared CAR-T cells and B16 cells were centrifuged at 500 g for 3 minutes, resuspended in 1640 medium (5% FBS), and sampled and counted; then, CAR-T cells and B16 melanoma cells were mixed into the same well according to the effector-target ratio (the ratio of the number of CAR-T cells and B16 cells) E:T = 1:1, 1:2, 1:4, and a duplicate well was set for each well; the control group was mixed with the same number of activated T cells and the same number of target cells into the same well; cultured at 37°C for 4 hours, and the results were analyzed by eBioscience TMAnnexin V apoptosis detection kit (Invitrogen, 88-8102-72) was used to detect the cells according to the instructions of the kit, and the percentage of Annexin V positive cells was compared; the results were as follows Fig. 9 As shown, the results showed that when the effector-target ratio E:T=1:1, 1:2 and 1:4, there were significant statistical differences between the two groups.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. An antibody targeting and recognizing mCD155, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and the light chain variable region comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3; characterized in that: It selects any of the following antibodies: (a) First antibody: the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 3; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 9, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 11; (b) Second antibody: the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 17, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 18, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 19; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 25, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO:

27.

2. The antibody targeting and recognizing mCD155 according to claim 1, characterized in that: In the first antibody, the heavy chain variable region further includes a heavy chain FR1 with a sequence as shown in SEQ ID NO:4 and / or a heavy chain FR2 with a sequence as shown in SEQ ID NO:5 and / or a heavy chain FR3 with a sequence as shown in SEQ ID NO:6 and / or a heavy chain FR4 with a sequence as shown in SEQ ID NO:7; and / or the light chain variable region further includes a light chain FR1 with a sequence as shown in SEQ ID NO:12 and / or a light chain FR2 with a sequence as shown in SEQ ID NO:13 and / or a light chain FR3 with a sequence as shown in SEQ ID NO:14 and / or a light chain FR4 with a sequence as shown in SEQ ID NO:15; or In the second antibody, the heavy chain variable region also includes a heavy chain FR1 with a sequence as shown in SEQ ID NO:20 and / or a heavy chain FR2 with a sequence as shown in SEQ ID NO:21 and / or a heavy chain FR3 with a sequence as shown in SEQ ID NO:22 and / or a heavy chain FR4 with a sequence as shown in SEQ ID NO:23; and / or the light chain variable region also includes a light chain FR1 with a sequence as shown in SEQ ID NO:28 and / or a light chain FR2 with a sequence as shown in SEQ ID NO:29 and / or a light chain FR3 with a sequence as shown in SEQ ID NO:30 and / or a light chain FR4 with a sequence as shown in SEQ ID NO:

31.

3. The antibody targeting and recognizing mCD155 according to claim 2, characterized in that: 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 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.

4. An antigen-binding fragment, characterized in that The invention comprises the antibody targeting and recognizing mCD155 as claimed in any one of claims 1 to 3.

5. The CAR structure targeting mCD155 is characterized in that: The invention comprises the antibody targeting and recognizing mCD155 as claimed in any one of claims 1 to 3 or the antigen-binding fragment as claimed in claim 4.

6. A nucleotide sequence characterized in that Encoding the antibody targeting and recognizing mCD155 as described in any one of claims 1 to 3, or the antigen binding fragment as described in claim 4, or the CAR structure as described in claim 5.

7. An expression vector, characterized in that: Comprising the nucleotide sequence of claim 6.

8. An engineered cell, characterized in that: Comprising the expression vector according to claim 7.

9. Use of the antibody targeting and recognizing mCD155 as described in any one of claims 1 to 3, the antigen binding fragment as described in claim 4, the CAR structure as described in claim 5, the nucleotide sequence as described in claim 6, the expression vector as described in claim 7, or the engineered cell as described in claim 8 in the preparation of a medicament for treating mCD155-mediated cancer.

10. Use of the antibody targeting and recognizing mCD155 according to any one of claims 1 to 3, the antigen-binding fragment according to claim 4, the nucleotide sequence according to claim 6, the expression vector according to claim 7, or the engineered cell according to claim 8 in the preparation of a product for qualitative and / or quantitative detection of mCD155.

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