CN113416253B - Isolated antigen ITPRIPL1 binding proteins and uses thereof - Google Patents

Abstract

The application discloses an isolated antigen ITPRIPL1 binding protein which can bind with an amino acid sequence shown as SEQ ID NO.25 in antigen ITPRIPL1. The isolated antigen ITPRIPL1 binding protein comprises HCDR1, HCDR2 and HCDR3 in a VH set forth in any one of amino acid sequences SEQ ID NO 5 or 14; and which comprises LCDR1, LCDR2 and LCDR3 in VL represented by any one of amino acid sequences SEQ ID NO 6 or 15.

Description

Isolated antigen ITPRIPL1 binding protein and use thereof

technical field

The application relates to the field of biomedicine, in particular to an isolated antigen ITPRIPL1 binding protein and its use.

Background technique

The protein encoded by ITPRIPL1 is inositol 1,4,5-trisphosphate receptor-interacting protein-like 1 (Inositol 1,4,5-trisphosphate receptor-interacting protein-like 1), and there is no report on the function of ITPRIPL1. According to the annotation of UniProtKB database, human ITPRIPL1 includes 555 amino acids, which are divided into extracellular region (1-103 amino acids), transmembrane region (104-124 amino acids), and intracellular region (125-555 amino acids).

T cells are key effector cells of the adaptive immune response and have many important roles in pathogen elimination and autoimmune diseases. There are several subpopulations of T cells, each with a different function. The TCR (T cell receptor) found on the surface of T cells is a heterodimer composed of alpha and beta polypeptide chains (constituting approximately 95% of the TCR population), or gamma and delta polypeptide chains. Each polypeptide comprises constant (C) and variable (V) regions. The constant region is anchored in the cell membrane, while the variable region extends extracellularly and is responsible for binding antigens. The cytoplasmic short tail of the TCR lacks the ability to transduce signals. Intracellular signaling is initiated by the CD3 protein complex, which contains the intracellular immunoreceptor tyrosine activation motif (ITAM).

The CD3 (cluster of differentiation 3) T-cell co-receptor is a protein complex consisting of four distinct chains. In mammals, the complex contains one CD3 gamma (γ) chain, one CD3 delta (δ) chain and two CD3 ε (ε) chains. These chains are associated with the TCR and the zeta chain (zeta chain), which generate activation signals in T lymphocytes. The TCR, zeta chain and CD3 molecule together form the TCR complex. The CD3γ, CD3δ and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily comprising a single extracellular immunoglobulin domain. TCRs cannot bind free epitopes/antigens, instead, TCRs bind enzymatically cleaved fragments of larger polypeptides associated with the major histocompatibility complex (MHC), which is synonymous with the human human leukocyte antigen (HLA) system . This interaction occurs in a space known as the immune synapse. MHC class I molecules are expressed on all nucleated cells in the body and present antigens to cytotoxic T cells on which CD8 stabilizes the MHC/TCR interaction. Activation of cytotoxic T cells subsequently leads to destruction of target cells. MHC class II is found on macrophages, B cells and dendritic cells. These immune cells present antigens to helper T cells with CD4 that stabilizes MHC/TCR interactions. Interactions between MHC class II and TCRs ultimately lead to antibody-mediated immune responses. Other co-stimulatory molecules, such as CD45, CD28 and CD2, contribute to T cell activation at the immune synapse and initiate the formation of TCR signalosomes, macromolecular protein complexes responsible for intracellular signaling. So far, there is no report on ITPRIPL1 as a ligand of CD3ε.

Axon guidance factor semaphorin is a large family with at least 20 members, all of which are secreted or membrane-bound proteins. As a protein of SEMA3 subgroup, SEMA3G is reported to bind NRP2, PlexinA4, PlexinD1, and can regulate the formation of lymphatic vessels and release reverse guidance signals to lymphatic endothelial cells. According to the annotations of the HumanProtein Atlas database, SEMA3G is mainly expressed on vascular endothelial cells and regulatory T cells (Treg) (Protein Sci.2018; 27:233-244).

Current studies have found that ITPRIPL1 protein is expressed in tumor cells and can act as a ligand to bind to CD3E and SEMA3G, thereby inhibiting the killing of tumor cells by T cells. Therefore, those skilled in the art are devoting themselves to developing an immunotherapy that can be used in combination with an ITPRIPL1-targeting antibody for tumors.

Contents of the invention

To achieve the above purpose, the present application provides an isolated antigen ITPRIPL1 binding protein, which can bind to the amino acid sequence shown in SEQ ID NO:25 in the antigen ITPRIPL1.

In certain embodiments, it comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region VH shown in any one of the amino acid sequences SEQ ID NO:5 and 14; and it comprises the amino acid sequences SEQ ID NO:6 and LCDR1, LCDR2 and LCDR3 in the light chain variable region VL shown in any one of 15.

In some embodiments, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 7, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 8, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 9.

In certain embodiments, wherein the amino acid sequence of the LCDR1 is as shown in any one of SEQ ID NO: 10 or 16, the amino acid sequence of the LCDR2 is KV, and the amino acid sequence of the LCDR3 is as SEQ ID NO : any of 11 or 17.

In certain embodiments, it comprises an antibody heavy chain constant region, and said antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

In certain embodiments, it comprises an antibody light chain constant region, and said antibody light chain constant region comprises a human Ig kappa constant region.

In certain embodiments, it comprises an antibody heavy chain HC, and said HC comprises the amino acid sequence set forth in any one of SEQ ID NO:3 or 12.

In certain embodiments, it comprises an antibody light chain LC, and the LC comprises the amino acid sequence set forth in any one of SEQ ID NO:4 or 13.

In certain embodiments, it comprises an antibody or antigen-binding fragment thereof, wherein said antigen-binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv and/or di- scFv.

In certain embodiments, the ITPRIPL1 protein comprises human ITPRIPL1 protein.

In some embodiments, the human ITPRIPL1 protein comprises the amino acid sequence shown in SEQ ID NO.1.

In another aspect, the present application also provides a chimeric antigen receptor comprising the isolated antigen ITPRIPL1 binding protein described in the present application.

In another aspect, the present application also provides an immunoconjugate comprising the isolated antigen ITPRIPL1 binding protein described in the present application.

In another aspect, the present application also provides one or more isolated nucleic acid molecules encoding the isolated antigen ITPRIPL1 binding protein or the chimeric antigen receptor described in the present application.

In another aspect, the present application also provides a vector comprising the nucleic acid molecule described in the present application.

On the other hand, the present application also provides a cell comprising the nucleic acid molecule or the vector described in the present application.

On the other hand, the present application also provides a pharmaceutical composition, which comprises the isolated antigen ITPRIPL1 binding protein described in the present application, the chimeric antigen receptor, the immunoconjugate, and optionally a pharmaceutical acceptable adjuvants.

On the other hand, the present application also provides a method for preparing the isolated antigen ITPRIPL1 binding protein described in the present application, the method comprising culturing the isolated antigen ITPRIPL1 binding protein described in the present application under the condition of expressing Apply to the cells as described.

On the other hand, the present application also provides the isolated antigen ITPRIPL1 binding protein described in the present application, the chimeric antigen receptor, the immunoconjugate, and/or the pharmaceutical composition described in the preparation Use in a medicament for preventing, alleviating and/or treating tumors.

In certain embodiments, the tumor comprises lymphoma.

The applicant creatively developed a targeting antibody that can bind to ITPRIPL1, which can bind to the above-mentioned target protein with high affinity and neutralize its function, and inhibit its binding to one or more ligands, so as to relieve the immune escape function of tumor cells , to promote the killing of tumor cells by immune cells in vivo and in vitro; the antibody provided by the application can be used as an active ingredient to prepare a drug for treating tumors, providing a new effective solution for tumor treatment. At the same time, the invention also provides the linear epitope corresponding to the antibody with excellent neutralizing function and the biomarker of the administered antibody.

The idea, specific structure and technical effects of the present application will be further described below in conjunction with the accompanying drawings, so as to fully understand the purpose, features and effects of the present application.

Description of drawings

Fig. 1 shows a graph of the specific binding between ITPRIPL1 and receptor protein CD3E or SEMA3G in the present application. Among them, Fig. 1A is the binding curve of ITPRIPL1 and CD3E, and Fig. 1B is the binding curve of ITPRIPL1 and SEMA3G.

Figure 2 shows the results of the inhibitory effect of ITPRIPL1 overexpressed cells and free ITPRIPL1 protein on PBMC killing tumor cells in the present application. Among them, Figure 2A is the gate setting without adding PBMC, Figure 2B is the verification of the gate when adding PBMC, Figure 2C is the wild-type HCT116 cells that were not stained with Annexin V and PI, Figure 2D is the double-stained Annexin V and PI PI wild-type HCT116 cells without PBMC, Figure 2E is the wild-type HCT116 cells with PBMC double-stained with Annexin V and PI, Figure 2F is double-stained with Annexin V and PI with ITPRIPL1 overexpression HCT116 cells without PBMC, Figure 2E 2G is ITPRIPL1-overexpressed HCT116 cells that were double-stained with Annexin V and PI and added to PBMCs. Figure 2H is double-stained with Annexin V and PI without adding ITPRIPL1 knockout HCT116 cells to PBMCs. Figure 2I is double-stained with Annexin V and PI and added to PBMCs ITPRIPL1 knockout HCT116 cells, Figure 2J shows the changes in PBMC killing rate under different ITPRIPL1 expression conditions.

Figure 3 shows the results of the binding of the mouse hybridoma antibody to ITPRIPL1 in the present application. Among them, Fig. 3A is the ELISA result graph of 1 microgram per milliliter of 100 hybridoma antibodies reacting with 1 microgram per milliliter ITPRIPL1, and Fig. 3B is the reaction of 1 microgram per milliliter of 9 strains of hybridoma antibodies reacting with 1 microgram per milliliter ITPRIPL1 Figure of ELISA results. Figure 3C is a graph showing the binding curve of 13B7 antibody to ITPRIPL1.

FIG. 4 shows the results of detecting the binding of each mouse hybridoma antibody to endogenous Jurkat cells highly expressing ITPRIPL1 by flow cytometry analysis technology in the present application. Among them, Figure 4A shows the gate setting of Jurkat cells, Figure 4B shows the statistical results of the binding rate of each hybridoma antibody, Figure 4C-4L shows different hybridoma antibodies 2E7, 5E5, 13B7, 13F7, 15C9, 16E1, 18B12, 18G5, Flow cytometric analysis results of 19B11 and 20E3 binding to Jurkat cells.

Fig. 5 shows the results of detecting the binding of the mouse hybridoma 13B7 antibody to various tumor cells expressing ITPRIPL1 by the flow cytometric analysis technique in the present application. Among them, Figure 5A is HCT116 without adding antibody control, Figure 5B is HCT116 adding antibody group, Figure 5C is A549 without adding antibody control, Figure 5D is A549 adding antibody group, Figure 5E is MC38 without adding antibody control, Figure 5F is MC38 adding Antibody group, Figure 5G is the MC38-ITPRIPL1 stable transfection without adding the antibody control, Figure 5H is the MC38-ITPRIPL1 stable transfection adding the antibody group, Figure 5I is the Jurkat without adding the antibody control, Figure 5J is Jurkat adding the antibody group, and Figure 5K is Raji was not added to the antibody control, Figure 5L is the Raji added antibody group, and Figure 5M is the statistical result graph of the binding rate of 13B7 antibody to different tumor cells expressing ITPRIPL1.

Fig. 6 shows the results of the flow cytometric analysis technique in the present application detecting the binding results of different concentrations of the mouse hybridoma 13B7 antibody and endogenous Jurkat cells highly expressing ITPRIPL1. Among them, Fig. 6A is the gate setting, Fig. 6B is the negative control, Fig. 6C-Fig. 6H are respectively the binding rate of 0.0625/0.125/0.25/0.5/1/2 microgram per milliliter of 13B7 antibody binding, and Fig. 6I is each The statistical results of the group combination rate.

Fig. 7 shows the results of Western blot analysis of the binding of 13B7 antibody to ITPRIPL1 in this application. Among them, Jurkat cells endogenously expressing ITPRIPL1, HCT116 cells endogenously expressing ITPRIPL1 and MC38 cells not expressing ITPRIPL1 were used for Western Blot experiments, and 13B7 antibody was used for incubation.

Figure 8 shows the results of blocking the binding of ITPRIPL1 to different proteins by different mouse hybridoma antibodies in the present application. Among them, FIG. 8A shows the results of blocking the binding of ITPRIPL1 and CD3E by different mouse hybridoma antibodies, and FIG. 8B shows the results of blocking the binding of ITPRIPL1 and SEMA3G by different mouse hybridoma antibodies.

Figure 9 shows the ELISA results of the binding of the mouse hybridoma monoclonal antibody to ITPRIPL1 in the present application.

FIG. 10 shows the results of detecting the binding of different mouse hybridoma monoclonal antibodies to endogenous Jurkat cells highly expressing ITPRIPL1 by flow cytometric analysis technology in the present application.

Figure 11 shows the statistical results of blocking the binding of ITPRIPL1 to different proteins by different mouse hybridoma monoclonal antibodies in the present application. Among them, Fig. 11A shows the results of different mouse hybridoma monoclonal antibodies blocking the combination of ITPRIPL1 and CD3E, and Fig. 11B shows the statistical graph of different mouse hybridoma antibodies blocking the combination of ITPRIPL1 and SEMA3G.

Figure 12 shows the results of identification of the antigen-binding region of ITPRIPL1 in this application. Among them, Figures 12A-12M are sequentially the statistical results of the binding of antibodies 18B12, 18B12D1A6, 13B7, 13B7A6H3, 16E1, 18G5, 20E3, 16E1D8H1, 5E5, 2E7, 19B7, 13F7, 18G5F3F4 to different peptides from ITPRIPL1 protein.

Fig. 13 shows the results of flow cytometry analysis of different mouse hybridoma monoclonal antibodies in the present application to promote the killing of Raji cells endogenously expressing ITPRIPL1 by PBMC. Among them, Figure 13A-B is the gate setting, Figure 13C is the Raji cell apoptosis control, Figure 13D is the killing situation of adding PBMC and negative serum, Figure 13E-Figure 13N is adding 0.5 micrograms per milliliter of PBMC respectively 13B7A6H3 mAb, 2 μg/ml 13B7A6H3 mAb, 0.5 μg/ml 16E1D8C4 mAb, 2 μg/ml 16E1D8C4 mAb, 0.5 μg/ml 18G5F3E5 mAb, 2 μg/ml The detection results of 18G5F3E5 monoclonal antibody, 0.5 micrograms per milliliter of 18B12D1 monoclonal antibody, 2 micrograms per milliliter of 18B12D1 monoclonal antibody, 0.5 micrograms per milliliter of 18B12D1A6 monoclonal antibody, and 2 micrograms per milliliter of 18B12D1A6 monoclonal antibody.

Fig. 14 shows the statistical chart of the cell flow cytometry detection results of different mouse hybridoma monoclonal antibodies in the present application promoting the killing of Raji cells endogenously expressing ITPRIPL1 by PBMC.

detailed description

In the present application, unless otherwise stated, the scientific and technical terms used in the present application have the meanings commonly understood by those skilled in the art. Moreover, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation steps used herein are all terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

In this application, the term "isolated" generally refers to artificially obtained or artificially synthesized from a natural state. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of. The term "isolated" does not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.

In this application, the term "antibody" generally refers to an immunoglobulin or fragment or derivative thereof, encompassing any polypeptide that includes an antigen combining site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , mutated and transplanted antibodies. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. For example, an IgM antibody consists of 5 basic heterotetrameric units plus another polypeptide called the J chain, while an IgA antibody consists of 2-5 basic 4-chain units that can combine with the J chain to form multivalent assemblies. For IgG, the 4-chain unit is typically about 150,000 Daltons. Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has spaced intrachain disulfide bridges. Each H chain has a variable domain/region (VH) at the N-terminus, followed by three constant domains/regions (CH) for each of the α and γ chains, followed by With four CH domains. Each L chain has a variable domain/region (VL) at its N-terminus and a constant domain/region (CL) at its other end. VL corresponds to VH and CL corresponds to the first constant domain (CH1) of the heavy chain.

In this application, the term "light chain variable region" generally refers to the amino-terminal domain of an antibody light chain. The light chain variable region is referred to as "VL". These domains are usually the most variable parts of an antibody's light chain (relative to other antibodies of the same class), and may include complementarity determining regions (CDRs) or hypervariable regions (HVRs) and framework regions (FRs).

In this application, the term "heavy chain variable region" generally refers to the amino-terminal domain of an antibody heavy chain. The heavy chain variable region is referred to as "VH". These domains are usually the most variable parts of an antibody's heavy chain (relative to other antibodies of the same class), and may include complementarity determining regions (CDRs) or hypervariable regions (HVRs) and framework regions (FRs).

In this application, the term "complementarity determining region" or the term "CDR" generally refers to the variable regions that together define the binding site of an antigen binding protein (e.g., native immunoglobulin, chimeric or humanized antibody) Amino acid sequences for binding affinity and specificity (see, e.g., Chothia et al., J. Mol. Biol. 196:901-917 (1987); Kabat et al., U.S. Dept of Health and Human Services NIH Publication ) No. 913242 (1991)). Typically, antibodies comprise six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3). Naturally occurring camelid antibodies, consisting only of heavy chains, remain functional and stable in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996). The boundaries of the CDRs of the variable region of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems are different. For example, the residue ranges defined by different assignment systems for CDR regions using Kabat and Chothia numbering may be different. Thus, where reference is made to defining antibodies with a particular CDR sequence as defined in the present invention, the scope of said antibody also covers antibodies whose variable region sequences comprise said particular CDR sequence, but due to the application of a different protocol (e.g. Different assignment system rules or combinations) result in the claimed CDR boundaries being different from the specific CDR sequences provided in this application. The CDRs of the antibodies of the present application can be manually assessed and determined according to any scheme in the art or a combination thereof. Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" covers those determined in any of the above ways. CDR sequences.

In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can be expressed by transforming, transducing or transfecting the host cell, so that the genetic material elements carried by it can be expressed in the host cell. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); phage such as lambda phage or M13 phage and animal viruses.

In this application, the term "cell" may generally be or have been a recipient of a nucleic acid molecule or vector, a single cell, cell line or cell culture. A cell may comprise a nucleic acid molecule as described herein or a vector as described herein. Cells can include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell. Cells may include cells transfected in vitro with the vectors described herein.

In this application, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, eg a human patient. For example, the pharmaceutical composition described in the present application may comprise the isolated antigen-binding protein described in the present application, the carrier described in the present application and/or the cell described in the present application, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also comprise one or more suitable (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. preparations. For example, acceptable ingredients of the composition are nontoxic to recipients at the dosages and concentrations employed. Pharmaceutical compositions of the present application include, but are not limited to, liquid, frozen and lyophilized compositions.

In this application, the term "pharmaceutically acceptable adjuvant" generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc. that are compatible with drug administration. Generally safe, nontoxic, and neither biologically nor otherwise undesirable.

The application will be further described below in conjunction with the examples. It should be understood that these examples are only for the purpose of illustration. The application can be embodied through many different forms of embodiments. example.

Example 1 Detection of ITPRIPL1 binding to receptor protein CD3E or SEMA3G

Enzyme-linked immunosorbent assay (ELISA) confirmed the concentration-dependent direct binding of ITPRIPL1 protein to CD3ε extracellular domain protein and SEMA3G extracellular domain protein, using ELISA special plate (costar, ME, USA). First, 0.03125/0.0625/0.125/0.25/0.5/1/2 micrograms per milliliter of ITPRIPL1 recombinant protein (cusabio, Wuhan, China) or 2 micrograms per milliliter of SEMA3G protein (cusabio, Wuhan, China) were dissolved in 100 microliters ELISA coating solution (Solarbio, Beijing, China) was used to coat the plate, and the negative control was coated with 100 microliters of coating solution without protein. Four times wrapped overnight. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37 degrees for 90 minutes. After washing with PBST, use 1 microgram per milliliter of CD3ε extracellular region protein fragment (Shenzhou, Beijing, China) with different concentrations of ITPRIPL1 protein or 0.03125/0.0625/0.125/0.25/0.5/1/2 microgram per milliliter The ITPRIPL1 recombinant protein was incubated with the same concentration of SEMA3G protein, and the CD3ε protein and ITPRIPL1 protein both had hFc tags, and the incubator was combined at 37 degrees for 60 minutes. After washing with PBST, specific anti-human Fc fragment antibody (Abcam, MA, USA) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, prepare chromogenic solution (Invitrogen, CA, USA), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and place in Color reading at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Figure 1 shows the results of the ELISA experiment. In the figure, the OD450 readings of the negative control after subtracting the blank coating, blocking and adding the corresponding ligand protein for each positive group after color development. 1A is the binding curve of ITPRIPL1 and CD3E, and FIG. 1B is the binding curve of ITPRIPL1 and SEMA3G. Figure 1A shows that different concentrations of ITPRIPL1 combined with CD3E (1ug/mL) increased with the concentration of ITPRIPL1, and Figure 1B showed that the ability of ITPRIPL1 (2ug/mL) to bind to different concentrations of SEMA3G increased with the increase of the concentration of SENA3G The increase indicates that ITPRIPL1 has the ability to specifically bind to receptor proteins CD3E and SEMA3G. The results indicated that the purified ITPRIPL1 protein had a concentration-dependent direct binding to the purified protein of the extracellular region of CD3 and the protein of the extracellular region of SEMA3G, respectively.

Example 2: Overexpression of ITPRIPL1 significantly reduces the killing of tumor cells by human peripheral blood mononuclear cells

1. Construction of HCT116-ITPRIPL1 stable transfection lines and HCT116-ITPRIPL1 knockout cells

The constructed full-length ITPRIPL1-Flag plasmid (shown in SEQ ID NO.1) and the empty pcDNA3.1 plasmid were transfected into HCT116 cells (ATCC, VA, USA), and cultured in an incubator for 24-48 hours , adding 1000 micrograms per milliliter of Geneticin (G418) (Gibco, CA, USA) for selection, after 10-14 days until all the cells in the non-transfected group died, HCT116-ITPRIPL1 stable transfected cells were obtained.

Place HCT116 cells (ATCC, VA, U.S.) in a 24-well plate (Corning, NY, U.S.) at an appropriate density (grow to a density of about 30-40% on the second day), and digest and count the next day. The GFP control lentivirus (Jiman, Shanghai, China) was used to infect the cells with a quantitative gradient, the medium was changed after 48 hours, and GFP fluorescence was observed under the microscope after 72 hours to determine the optimal virus-to-cell infection ratio and explore the MOI value. After determining the MOI value, re-plate, infect HCT116 cells with the Cas9 system lentivirus containing blasticidin resistance (Jiman, Shanghai, China) at the MOI value, change the medium after 48 hours, and add concentration gradient after 72 hours Blasticidin (Invivogen, CA, USA) was used for screening for 10-14 days, and the cell line obtained by final screening was HCT116 cells containing the Cas9 system. Then, HCT116 cells containing the Cas9 system were plated and counted in a 24-well plate, and infected with a lentivirus containing puromycin-resistant sgRNA that specifically cuts ITPRIPL1 at an MOI value, and the medium was changed after 48 hours, and the concentration gradient was entered after 72 hours Puromycin (Invivogen, CA, USA) was selected for 10-14 days to obtain the HCT116-ITPRIPL1 knockout cell line.

2. Flow cytometry proved that overexpression of ITPRIPL1 can inhibit the killing of tumor cells by PBMC. Knockdown of ITPRIPL1 expressed by tumor cells can significantly increase the killing

The revived PBMC cells were activated with 1 ug/mL Anti-CD3/CD28 (Invitrogen, CA, USA) 24 hours in advance. Count the PBMC cells, HCT116-ITPRIPL1 stable transfection cells and HCT116-ITPRIPL1 knockout cells, respectively adjust the cell number to 1×10 ⁶ /ml, add 100 microliters to a 96-well plate (Thermo Fisher, MA, USA), After mixing, place in the incubator and incubate for 6 hours. The 96-well plate was taken out, the cells in each well were placed in EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. The CD45-APC antibody (Invitrogen, CA, USA) was diluted 1:20 with cell staining buffer, and 200 microliters of the mixture was added to each EP tube to resuspend, and incubated at room temperature for 30 minutes. After centrifugation at 400 rcf for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 1 ml of binding buffer (Beyond, Shanghai, China); the centrifugation and washing were repeated. Set up each experimental group and control group (the self-apoptotic group without PBMC as the control group, and the cells in each group with PBMC as the experimental group), according to the conditions, add 100 microliters of binding buffer, 5 microliters Annexin V-FITC (Biyuntian, Shanghai, China) and 10 μl PI (Biyuntian, Shanghai, China) were incubated at room temperature for 15 minutes, and then 400 μl of binding buffer were added to each, and transferred to flow tubes (Falcon, NY, The results are shown in Figure 2. The results show that overexpression of ITPRIPL1 inhibits the killing effect of PBMC on tumor cells, while knocking out ITPRIPL1 removes the inhibitory effect.

Example 3: Mouse hybridoma antibodies can specifically bind to ITPRIPL1

1. Preparation of ITPRIPL1 mouse hybridoma antibody

(1) Human ITPRIPL1-Fc recombinant protein (as shown in SEQ ID NO.2) was used as an immunogen to immunize C57BL/6 mice, and multiple immunizations were performed to enhance the effect:

1) For the first immunization, antigen 50 μg/monkey, plus Freund's complete adjuvant subcutaneous injection in multiple points, with an interval of 3 weeks;

2) For the second immunization, the dosage route is the same as above, with Freund's incomplete adjuvant added, and the interval is 3 weeks;

3) For the third immunization, the dose is the same as above, without adjuvant, and the interval of intraperitoneal injection is 3 weeks;

4) Booster immunization, dose 50 μg, intraperitoneal injection. Blood was collected 3 days after the last injection to measure its potency.

(2) After detecting that the immune effect meets the requirements, blood is collected multiple times, polyclonal antibody is separated, and polyclonal antibody is purified. The specific experimental steps include:

1) Prepare protein G sepharose CL-4B affinity column. Prepare 10mL of protein G sepharose CL-4B filler, mix equal volumes of filler and TBS buffer solution in a vacuum bottle, and stir. Vacuum for 15 minutes to remove air bubbles in the filling. Slowly add protein G sepharose CL-4B filler into the glass column, use the pump to control the filling speed to 1mL/min-2mL/min, avoid column drying, and use 10 times the bed volume and pre-cooled TBS buffer solution to balance the column;

2) Preparation of polyclonal antibodies. Thaw the polyclonal antibody slowly in ice water or in a 4°C refrigerator to avoid protein aggregation. Add solid sodium azide to a concentration of 0.05%, centrifuge at 15,000×g for 5 minutes at 4°C, remove the clarified polyclonal antibody and filter through a filter to remove excess fat;

3) Affinity chromatography. Dilute the antibody with TBS buffer solution at a ratio of 1:5, and then filter through a filter. Put the polyclonal antibody onto the column at a rate of 0.5 mL per minute. In order to ensure the combination of the polyclonal antibody and the filler, it is necessary to load the column twice continuously and keep the sample effluent. Wash the column with TBS buffer solution until Aλ280nm<0.008, then add Ph 2.7 elution buffer solution, and elute at a speed of 0.5mL/min until all proteins flow down. Use a 1.5mL EP tube that has been added with 100 μL of neutralizing buffer solution to collect the eluate in separate tubes. After mixing, check the pH of the eluent with pH test paper. When the pH is lower than 7, use the neutralizing buffer to adjust to about pH 7.4 to prevent Antibody denaturation. Add 10 mL of elution buffer solution with pH 1.9 to the column, and collect the eluate according to the above method until Aλ280nm<0.008. The protein content in each tube was determined using a spectrophotometer.

2. Enzyme-linked immunosorbent assay (ELISA) verification of the binding of each hybridoma antibody to ITPRIPL1

In order to verify the binding ability of each hybridoma antibody to ITPRIPL1, enzyme-linked immunosorbent assay (ELISA) was used to confirm the direct binding of each antibody to ITPRIPL1 protein. A special plate for ELISA (costar, ME, USA) was used. First, 100 microliters (1 microgram per milliliter) of ITPRIPL1 recombinant protein (cusabio, Wuhan, China) ELISA coating solution (Solarbio, Beijing, China) was used to coat the plate, and the negative control was coated with 100 microliters of TPRIPL1 recombinant protein. Liquid cladding board. Four times wrapped overnight. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37° C. for 90 minutes. After washing with PBST, bind in an incubator at 37°C for 60 minutes. After washing with PBST, specific anti-mouse Fc fragment antibody (Consun, Shanghai, China) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, prepare chromogenic solution (Invitrogen, CA, USA), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and place in Color reading at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Figure 3 is the result of the ELISA experiment. The upper left figure shows the ELISA results of the binding of all 100 obtained antibodies to ITPRIPL1 protein, and the upper right figure shows the results of a separate repeated experiment of 9 antibodies with better binding selected from the results in the upper left figure. The figure below shows the concentration-dependent binding curve of 13B7 antibody. Experiments showed that the binding ability of each antibody to ITPRIPL1 was different, indicating the heterogeneity among antibodies, and 13B7 was the antibody with the strongest binding ability. The above results demonstrate that each hybridoma can bind to ITPRIPL1 with different binding abilities.

2. Flow cytometry (FACS) verification of the binding of each hybridoma antibody to ITPRIPL1

In order to further verify the binding ability of each hybridoma antibody to ITPRIPL1, flow cytometry (FACS) was used to confirm the direct binding of each antibody to ITPRIPL1 protein. Count the Jurkat cells (ATCC, VA, U.S.) that endogenously highly express ITPRIPL1, adjust the cell number to 1×10 ⁶ /ml, add 100 microliters to each 96-well plate (Thermo Fisher, MA, U.S.), add to each The hybridoma antibody or control serum at a final concentration of 1 microgram per milliliter was added to the wells, and incubated in an incubator for 30 minutes. The 96-well plate was taken out, the cells in each well were placed in EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. Anti-mouse Fc segment-Alexa Fluor488 antibody (Invitrogen, CA, USA) was diluted 1:500 with cell staining buffer, 200 microliters of the mixture was added to each EP tube to resuspend, and incubated at room temperature for 30 minutes. After centrifugation at 400rcf for 5 minutes, discard the supernatant, resuspend and wash the cells with 1 ml of cell staining buffer; repeat the centrifugation and washing. Add 300 microliters of cell staining buffer to each, transfer to a flow tube (Falcon, NY, USA) and test on a machine (Miltenyi, Cologne, Germany).

Fig. 4 is the result of the FACS experiment, which shows that the binding ability of each antibody to ITPRIPL1 is different, showing the heterogeneity among antibodies, among which 13B7 is the antibody with the strongest binding ability.

Example 4 13B7 antibody can specifically bind ITPRIPL1 with high affinity

1. Flow cytometry (FACS) to verify the binding of 13B7 antibody to cells with different expression levels of ITPRIPL1

In order to further verify the binding ability of 13B7 to ITPRIPL1, flow cytometry (FACS) was used to confirm that the 13B7 antibody directly binds to various types of cells with different expression levels of ITPRIPL1. Cells with different expression levels of endogenous ITPRIPL1, namely HCT116 (ATCC, VA, USA), A549 (ATCC, VA, USA), MC38 (Kerafast, MA, USA), Jurkat (ATCC, VA, USA), Raji cells (ATCC, VA, U.S.) and MC38-ITPRIPL1 stable transfection strain cell count, adjust the cell number to be 1x10 ⁶ / milliliter, each add 100 microliters to the 96-well plate (Thermo Fisher, MA, U.S.), add to each well Add 13B7 antibody at a final concentration of 1 microgram per milliliter, and incubate in the incubator for 30 minutes. The 96-well plate was taken out, the cells in each well were placed in EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. Anti-mouse Fc fragment-AlexaFluor 488 antibody (Invitrogen, CA, USA) was diluted 1:500 with cell staining buffer, 200 microliters of the mixture was added to each EP tube to resuspend, and incubated at room temperature for 30 minutes. After centrifugation at 400rcf for 5 minutes, discard the supernatant, resuspend and wash the cells with 1 ml of cell staining buffer; repeat the centrifugation and washing. Add 300 microliters of cell staining buffer to each, transfer to a flow tube (Falcon, NY, USA) and test on a machine (Miltenyi, Cologne, Germany).

Figure 5 shows the results of the FACS experiment. The results of the experiment show that the binding force between each cell line and the 13B7 antibody is basically consistent with the expression of ITPRIPL1 protein in each cell line, indicating that the 13B7 antibody can specifically bind to ITPRIPL1 on the cell line expressing ITPRIPL1. combined.

2. Flow cytometry (FACS) verified that 13B7 antibody can bind Jurkat cells in a concentration-dependent manner

To verify the binding ability of 13B7 to ITPRIPL1, flow cytometry (FACS) was used to verify the concentration-dependent binding of 13B7 antibody to Jurkat cells. Count the Jurkat cells endogenously highly expressing ITPRIPL1, adjust the cell number to 1×10 ⁶ /ml, add 100 microliters to a 96-well plate (Thermo Fisher, MA, USA), and add a final concentration of 0.0625/0.125/0.25/0.5/1/2 micrograms per milliliter of 13B7 antibody, incubated in the incubator for 30 minutes. The 96-well plate was taken out, the cells in each well were placed in EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. Anti-mouse Fc fragment-Alexa Fluor 488 antibody (Invitrogen, CA, USA) was diluted 1:500 with cell staining buffer, 200 microliters of the mixture was added to each EP tube to resuspend, and incubated at room temperature for 30 minutes. After centrifugation at 400rcf for 5 minutes, discard the supernatant, resuspend and wash the cells with 1 ml of cell staining buffer; repeat the centrifugation and washing. Add 300 microliters of cell staining buffer to each, transfer to a flow tube (Falcon, NY, USA) and test on a machine (Miltenyi, Cologne, Germany).

Fig. 6 is the result of the FACS experiment, which shows that Jurkat cells can bind the 13B7 antibody with high affinity in a concentration-dependent manner.

3. Western blot method to verify that the 13B7 antibody can specifically bind to the ITPRIPL1 protein of cells with different ITPRIPL1 expression levels

The Jurkat, HCT116, and MC38 cells containing endogenously expressed ITPRIPL1 were counted, and 1×10 ⁶ cells were taken out, washed with PBS and centrifuged, and then mixed with 60 microliters of RIPA lysate (Beiyuntian, Shanghai, China) and 1 : 100% protease inhibitor, phosphatase inhibitor, PMSF triple (Consun, Shanghai, China) mixed lysate lysed on ice for 15 minutes, took liquid nitrogen for three freezing and three thawing, after centrifugation, loading buffer (Biyuntian, Shanghai, China) to prepare input protein samples after mixing and denaturation in a metal bath at 100°C. Then configure 12.5% PAGE gel (Yase, Shanghai, China) in the gel plate (Bole, CA, USA) according to the instructions, put the prepared gel in the electrophoresis tank (Bole, CA, USA), and connect the power supply (Bole, CA, U.S.) Let the strips run through the stacking gel with a constant voltage of 80 volts, and run through the separating gel with a constant voltage of 120 volts. When the strips reached the bottom of the separating gel, transfer to the membrane using a wet method in a transfer tank (Bio-Rad, CA, USA) with a constant current of 350 mA for 90 minutes. After the membrane transfer was completed, the membrane was sheared according to the mass of the ITPRIPL1 protein and the internal reference GAPDH protein. Block with fast blocking solution (Yase, Shanghai, China) for ten minutes, dilute with 13B7 antibody at a final concentration of 1 microgram per milliliter and incubate the corresponding bands of ITPRIPL1 and GAPDH with GAPDH-HRP antibody (Conson, Shanghai, China), 4°C overnight. The next day, after washing with TBST, incubate with specific anti-mouse secondary antibody (Consun, Shanghai, China) diluted in 5% skimmed milk powder (Sangon, Shanghai, China) dissolved in TBS for one hour at room temperature, wash with TBST, and place Exposure to a gel imager (Bio-Rad, CA, USA) for one minute in a mixed luminescence solution (San Er, Shanghai, China).

Fig. 7 is the result of the Western Blot experiment. As shown in the figure, according to the GAPDH internal reference, the 13B7 antibody can specifically display a band at the molecular weight position of the ITPRIPL1 protein, which is consistent with the endogenous expression of ITPRIPL1 in each cell, indicating that the 13B7 antibody can specifically bind to the ITPRIPL1 protein to combine.

Example 5 Determination of the blocking effect of each hybridoma antibody on the combination of ITPRIPL1 and CD3E/SEMA3G

In order to verify whether each hybridoma antibody has a blocking effect on the binding of ITPRIPL1 to CD3E or SEMA3G, an enzyme-linked immunosorbent assay (ELISA) was used to verify the blocking effect of each antibody. A special plate for ELISA (costar, ME, USA) was used. First, 1 microgram per milliliter of ITPRIPL1-Fc-tagged recombinant protein (Abclonal, Wuhan, China) or 1 microgram per milliliter of CD3E-Fc-labeled protein (Acro, Beijing, China) was dissolved in 100 microliters of ELISA coating solution (Solarbio, China). Beijing, China) and the negative control was coated with 100 microliters of coating solution without protein. Four times wrapped overnight. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37 degrees for 90 minutes. After washing with PBST, SEMA3G-His tagged protein (cusabio, Wuhan, China) with a final concentration of 1 microgram per milliliter was added to the wells coated with ITPRIPL1-Fc protein, and a hybridoma antibody with a final concentration of 1/2 microgram per milliliter was added at the same time; Add ITPRIPL1-His tagged protein (cusabio, Wuhan, China) at a final concentration of 1 microgram per milliliter to the wells coated with CD3E-Fc protein, and at the same time add hybridoma antibody at a final concentration of 1/2 microgram per milliliter; incubator at 37 degrees Combine for 60 minutes. After washing with PBST, specific anti-His fragment antibody (Abcam, MA, USA) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, prepare chromogenic solution (Invitrogen, CA, USA), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and place in Color reading at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Figure 8 is the result of the ELISA experiment. The upper figure shows the blocking condition of each hybridoma antibody when ITPRIPL1-CD3E is combined, and the lower figure shows the blocking condition of each hybridoma antibody when ITPRIPL1-SEMA3G is combined. The experimental results showed that each hybridoma antibody could block the binding of ITPRIPL1 to CD3E or SEMA3G to varying degrees, showing the heterogeneity among the antibodies, among which the 18B12 and 13B7 antibodies had the best binding effects.

Example 6 Monoclonal antibody can bind ITPRIPL1 with higher affinity

1. Immunosorbent assay (ELISA) to verify the binding ability of each monoclonal antibody to ITPRIPL1

After monoclonal purification of each hybridoma antibody, 16 monoclonal antibodies with better growth conditions were obtained. In order to verify the binding ability of each monoclonal antibody to ITPRIPL1, enzyme-linked immunosorbent assay (ELISA) was used to verify the direct binding of each antibody to ITPRIPL1 protein. A special plate for ELISA (costar, ME, USA) was used. First, 100 microliters of 1 microgram per milliliter ITPRIPL1 recombinant protein (cusabio, Wuhan, China) ELISA coating solution (Solarbio, Beijing, China) was used to coat the plate, and the negative control was coated with 100 microliters of protein-free coating solution. Four times wrapped overnight. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37 degrees for 90 minutes. After washing with PBST, different monoclonal antibodies were added with a final concentration of 1 microgram per milliliter, and combined in an incubator at 37 degrees for 60 minutes. After washing with PBST, specific anti-mouse Fc fragment antibody (Consun, Shanghai, China) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, prepare chromogenic solution (Invitrogen, CA, USA), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and place in Color reading at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Fig. 9 is the result of the ELISA experiment. The experimental results show that there is a certain gap in the binding ability of each monoclonal antibody to ITPRIPL1, and the overall binding ability to ITPRIPL1 is significantly stronger than that before monoclonal purification, indicating that the monoclonal antibody can bind ITPRIPL1 with a higher affinity.

2. Flow cytometry (FACS) to verify the binding of each monoclonal antibody to ITPRIPL1

In order to further verify the binding ability of each monoclonal antibody to ITPRIPL1, flow cytometry (FACS) was used to confirm the direct binding of each antibody to ITPRIPL1 protein. Count the Jurkat cells endogenously highly expressing ITPRIPL1, adjust the cell number to 1×10 ⁶ /ml, add 100 microliters to a 96-well plate (Thermo Fisher, MA, USA), and add a final concentration of 1 microgram per milliliter of monoclonal antibody or control serum, incubate for 30 minutes in the incubator. The 96-well plate was taken out, the cells in each well were placed in EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. Anti-mouse Fc fragment-Alexa Fluor 488 antibody (Invitrogen, CA, USA) was diluted 1:500 with cell staining buffer, 200 microliters of the mixture was added to each EP tube to resuspend, and incubated at room temperature for 30 minutes. After centrifugation at 400rcf for 5 minutes, discard the supernatant, resuspend and wash the cells with 1 ml of cell staining buffer; repeat the centrifugation and washing. Add 300 microliters of cell staining buffer to each, transfer to a flow tube (Falcon, NY, USA) and test on a machine (Miltenyi, Cologne, Germany).

The results are shown in Figure 10. The binding ability of each monoclonal antibody to ITPRIPL1 is different, showing the heterogeneity among antibodies, and the overall binding ability is stronger than that before monoclonal purification. The above results prove that each hybridoma can bind to ITPRIPL1 protein with different binding ability, and the binding ability to ITPRIPL1 is stronger after monoclonal purification.

Example 7: Determination of the blocking effect of each monoclonal antibody on the binding of ITPRIPL1 to CD3E/SEMA3G

In order to verify whether the obtained monoclonal antibody has a blocking effect on the binding of ITPRIPL1 to CD3E or SEMA3G, enzyme-linked immunosorbent assay (ELISA) was used to verify the blocking effect of each antibody. A special plate for ELISA (costar, ME, USA) was used. First, ITPRIPL1-Fc-tagged recombinant protein (Abclonal, Wuhan, China) at a final concentration of 1 μg/ml or CD3E-Fc-tagged protein (Acro, Beijing, China) at a final concentration of 1 μg/ml was dissolved in 100 μl ELISA The plate was coated with coating solution (Solarbio, Beijing, China), and the negative control was coated with 100 microliters of coating solution without protein. Coating overnight at 4°C. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37 degrees for 90 minutes. After washing with PBST, SEMA3G-His tagged protein (cusabio, Wuhan, China) with a final concentration of 1 microgram per milliliter was added to the wells coated with ITPRIPL1-Fc protein, and a monoclonal antibody with a final concentration of 1 microgram per milliliter was added at the same time; CD3E-Fc protein wells were added with a final concentration of 1 microgram per milliliter of ITPRIPL1-His tagged protein (cusabio, Wuhan, China), while adding a final concentration of 1/2 microgram per milliliter of monoclonal antibody; minute. After washing with PBST, specific anti-His fragment antibody (Abcam, MA, USA) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, prepare chromogenic solution (Invitrogen, CA, USA), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and place in Color reading at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Figure 11 is the result of the ELISA experiment. The experimental results showed that each monoclonal antibody still has some differences in the blocking effect of ITPRIPL1 on CD3E/SEMA3G, and the blocking effect of 13B7A6H3/18B12D1A6/18B12D1F7 is better, which is consistent with the conclusion before monoclonal purification.

Example 8 Hybridoma Antibody, Monoclonal Antibody, and Polypeptide Fragment Binding Determination

Based on the protein sequence of the extracellular segment of ITPRIPL1, a total of 17 segments were designed with a 1/3 overlapping sequence (GenScript, Nanjing, China). Its sequence is shown in SEQ ID NO.18-34. The resulting polypeptide fragments were dissolved in DMSO at a final concentration of 400 micrograms per milliliter, enzyme-linked immunosorbent assay (ELISA) was used to verify the antibodies of each hybridoma, and 4 monoclonal antibodies were selected to verify the binding of the polypeptide fragments. A special plate for ELISA (costar, ME, USA) was used. Firstly, the polypeptide segment with a final concentration of 1 microgram per milliliter was coated with 100 microliters of ELISA coating solution (Solarbio, Beijing, China), and the negative control was coated with 100 microliters of coating solution without protein. Four times wrapped overnight. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37 degrees for 90 minutes. After washing with PBST, different hybridoma antibodies or monoclonal antibodies with a final concentration of 1 microgram per milliliter were added, and combined in an incubator at 37 degrees for 60 minutes. After washing with PBST, specific anti-mouse Fc fragment antibody (Consun, Shanghai, China) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, prepare chromogenic solution (Invitrogen, CA, USA), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and place in Color reading at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Figure 12 is the result of the ELISA experiment. Reads that bind to a specific peptide fragment more than three times higher than background and other peptide fragments are considered to bind to a specific peptide fragment. The experimental results show that the monoclonal antibodies produced by 13B7/18B12 with better blocking effects all bind to P8, while the hybridoma antibodies and monoclonal antibodies with no or poor blocking effects have no specific binding peptides, indicating that P8 as the dominant binding site.

Example 9: Determination of the promotion effect of ITPRIPL1 monoclonal antibody on PBMC cells killing tumor cells

Flow cytometry proved that the monoclonal antibody that blocks the combination of ITPRIPL1 and CD3E/SEMA3G can promote the killing of tumor cells by PBMC.

The revived PBMC cells were activated with 1 ug/mL Anti-CD3/CD28 (Invitrogen, CA, USA) 24 hours in advance. Count the PBMC cells and Raji cells, adjust the cell number to 4x10 ⁶ /ml or 1x10 ⁶ /ml respectively, add 100 microliters to a 96-well plate (Thermo Fisher, MA, U.S.), and add two different concentrations of monoclonal antibody or control serum, mixed and placed in the incubator for co-incubation for 6 hours. The 96-well plate was taken out, the cells in each well were placed in EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. After centrifugation at 400 rcf for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 1 ml of binding buffer (Beyond, Shanghai, China); the centrifugation and washing were repeated. Set up each experiment and control group, add 100 microliters of binding buffer, 5 microliters of Annexin V-FITC (Beyond, Shanghai, China) and 10 microliters of PI (Beyond, Shanghai, China) according to the conditions, and incubate at room temperature for 15 minutes , and then add 400 microliters of binding buffer respectively, transfer to the flow tube (Falcon, NY, the United States) and test on the machine (Miltenyi, Cologne, Germany), the results are shown in Figures 13 and 14, the results show that the same as Compared with the control, among the antibodies, the monoclonal antibody 13B7A6H3/18B12D1A6, which has the effect of blocking the binding of ITPRIPL1 to CD3E/SEMA3G and can bind to P8 (SEQ ID NO.25), significantly increased the killing effect of PBMC on Raji cells.

According to sequencing, the heavy chain sequence of the monoclonal antibody 13B7A6H3 is shown in SEQ ID NO.3, the light chain sequence is shown in SEQ ID NO.4, and the heavy chain variable region VH sequence is shown in SEQ ID NO.5. The variable region VL of the light chain is shown in SEQ ID NO.6, the sequence of the complementary determining region HCDR1 of the heavy chain is shown in SEQ ID NO.7, the sequence of the complementary determining region HCDR2 of the heavy chain is shown in SEQ ID NO.8, and the sequence of the complementary determining region of the heavy chain is shown in SEQ ID NO.8. The sequence of the complementarity determining region HCDR3 is shown in SEQ ID NO.9, the sequence of the light chain complementarity determining region LCDR1 is shown in SEQ ID NO.10, the sequence of the light chain complementarity determining region LCDR2 is KV, and the sequence of the light chain complementarity determining region LCDR3 is KV. The sequence is shown in SEQ ID NO.11; the heavy chain sequence of monoclonal antibody 18B12D1A6 is shown in SEQ ID NO.12, the light chain sequence is shown in SEQ ID NO.13, and the heavy chain variable region VH sequence is shown in SEQ ID NO .14, the light chain variable region VL is shown in SEQ ID NO.15, the sequence of the heavy chain complementarity determining region HCDR1 is shown in SEQ ID NO.7, and the sequence of the heavy chain complementarity determining region HCDR2 is shown in SEQ ID NO.8 As shown, the sequence of the complementarity determining region HCDR3 of the heavy chain is shown in SEQ ID NO.9, the sequence of the complementarity determining region LCDR1 of the light chain is shown in SEQ ID NO.16, the sequence of the complementarity determining region LCDR2 of the light chain is KV, and the sequence of the complementarity determining region LCDR2 of the light chain is KV. The sequence of the complementarity determining region LCDR3 is shown in SEQ ID NO.17.

The preferred specific embodiments of the present application have been described in detail above. It should be understood that those skilled in the art can make many modifications and changes based on the concept of the present application without creative efforts. Therefore, all technical solutions that can be obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present application on the basis of the prior art shall be within the scope of protection defined by the claims.

sequence listing

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Met Glu Trp Arg Ile Phe Leu Phe Ile Leu Ser Gly Thr Ala Gly Val

1 5 10 15

His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro

20 25 30

Gly Ala Ser Val Arg Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr

35 40 45

Asp Tyr Val Ile Ser Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu

50 55 60

Trp Ile Gly Glu Ile Phe Pro Arg Thr Gly Ser Thr Tyr Tyr Asn Glu

65 70 75 80

Asn Phe Lys Ala Thr Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr

85 90 95

Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ala Tyr

100 105 110

Phe Cys Ala Phe Ile Thr Ser Val Asp Trp Ala Met Glu Tyr Trp Gly

115 120 125

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser

130 135 140

Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val

145 150 155 160

Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Thr Trp Asn Ser Gly Ser Leu Ser Ser Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro

195 200 205

Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro

210 215 220

Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly

225 230 235 240

Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile

245 250 255

Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys

260 265 270

Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln

275 280 285

Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln

290 295 300

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu

305 310 315 320

Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg

325 330 335

Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

340 345 350

Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro

355 360 365

Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr

370 375 380

Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln

385 390 395 400

Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly

405 410 415

Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu

420 425 430

Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn

435 440 445

His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

450 455 460

<210> 4

<211> 238

<212> PRT

<213> Artificial Sequence

<220>

<223> 13B7A6H3 antibody light chain LC

<400> 4

Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Gly

1 5 10 15

Ser Ser Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val

20 25 30

Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Glu Ser Leu

35 40 45

Val Asn Ser Lys Gly Asn Thr His Leu His Trp Tyr Leu Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys

100 105 110

Ser Gln Ser Thr His Ala Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro

130 135 140

Ser Ser Glu Gln Leu Thr Ser Ser Gly Gly Ala Ser Val Val Cys Phe Leu

145 150 155 160

Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly

165 170 175

Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp

195 200 205

Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr

210 215 220

Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

<210> 5

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> 13B7A6H3 heavy chain variable region VH

<400> 5

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Arg Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Val Ile Ser Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Phe Pro Arg Thr Gly Ser Thr Tyr Tyr Asn Glu Asn Phe

50 55 60

Lys Ala Thr Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ala Tyr Phe Cys

85 90 95

Ala Phe Ile Thr Ser Val Asp Trp Ala Met Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 6

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> 13B7A6H3 light chain variable region VL

<400> 6

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Glu Ser Leu Val Asn Ser

20 25 30

Lys Gly Asn Thr His Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Ala Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 7

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> 13B7A6H3/18B12D1A6 Antibody HCDR1

<400> 7

Gly Tyr Thr Phe Thr Asp Tyr Val

1 5

<210> 8

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> 13B7A6H3/18B12D1A6 Antibody HCDR2

<400> 8

Ile Phe Pro Arg Thr Gly Ser Thr

1 5

<210> 9

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> 13B7A6H3/18B12D1A6 antibody HCDR3

<400> 9

Ala Phe Ile Thr Ser Val Asp Trp Ala Met Glu Tyr

1 5 10

<210> 10

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> 13B7A6H3 Antibody LCDR1

<400> 10

Glu Ser Leu Val Asn Ser Lys Gly Asn Thr His

1 5 10

<210> 11

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> 13B7A6H3 Antibody LCDR3

<400> 11

Ser Gln Ser Thr His Ala Pro Tyr Thr

1 5

<210> 12

<211> 461

<212> PRT

<213> Artificial Sequence

<220>

<223> 18B12D1A6 antibody heavy chain HC

<400> 12

Met Glu Trp Arg Ile Phe Leu Phe Ile Leu Ser Gly Thr Ala Gly Val

1 5 10 15

His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro

20 25 30

Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

35 40 45

Asp Tyr Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu

50 55 60

Trp Ile Gly Glu Ile Phe Pro Arg Thr Gly Ser Thr Tyr Phe Asn Glu

65 70 75 80

Asn Phe Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr

85 90 95

Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ala Tyr

100 105 110

Phe Cys Ala Phe Ile Thr Ser Val Asp Trp Ala Met Glu Tyr Trp Gly

115 120 125

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser

130 135 140

Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val

145 150 155 160

Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Thr Trp Asn Ser Gly Ser Leu Ser Ser Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro

195 200 205

Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro

210 215 220

Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly

225 230 235 240

Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile

245 250 255

Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys

260 265 270

Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln

275 280 285

Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln

290 295 300

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu

305 310 315 320

Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg

325 330 335

Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

340 345 350

Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro

355 360 365

Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr

370 375 380

Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln

385 390 395 400

Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly

405 410 415

Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu

420 425 430

Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn

435 440 445

His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

450 455 460

<210> 13

<211> 238

<212> PRT

<213> Artificial Sequence

<220>

<223> 18B12D1A6 antibody light chain LC

<400> 13

Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala

1 5 10 15

Ser Ser Ser Asp Val Val Met Thr Gln Ile Pro Leu Ser Leu Pro Val

20 25 30

Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu

35 40 45

Ala Asn Ser Lys Gly Asn Thr His Leu His Trp Tyr Leu Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Phe Cys

100 105 110

Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro

130 135 140

Ser Ser Glu Gln Leu Thr Ser Ser Gly Gly Ala Ser Val Val Cys Phe Leu

145 150 155 160

Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly

165 170 175

Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp

195 200 205

Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr

210 215 220

Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

<210> 14

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> 18B12D1A6 heavy chain variable region VH

<400> 14

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Phe Pro Arg Thr Gly Ser Thr Tyr Phe Asn Glu Asn Phe

50 55 60

Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ala Tyr Phe Cys

85 90 95

Ala Phe Ile Thr Ser Val Asp Trp Ala Met Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 15

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> 18B12D1A6 light chain variable region VL

<400> 15

Asp Val Val Met Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Ala Asn Ser

20 25 30

Lys Gly Asn Thr His Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Ala

<210> 16

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> 18B12D1A6 Antibody LCDR1

<400> 16

Gln Ser Leu Ala Asn Ser Lys Gly Asn Thr His

1 5 10

<210> 17

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> 18B12D1A6 Antibody LCDR3

<400> 17

Ser Gln Ser Thr His Val Pro Tyr Thr

1 5

<210> 18

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P1

<400> 18

His Pro Leu Met Val Ser Asp Arg Met Asp Leu Asp Thr Leu Ala

1 5 10 15

<210> 19

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P2

<400> 19

Val Ser Asp Arg Met Asp Leu Asp Thr Leu Ala Arg Ser Arg Gln

1 5 10 15

<210> 20

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P3

<400> 20

Met Asp Leu Asp Thr Leu Ala Arg Ser Arg Gln Leu Glu Lys Arg

1 5 10 15

<210> 21

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P4

<400> 21

Thr Leu Ala Arg Ser Arg Gln Leu Glu Lys Arg Met Ser Glu Glu

1 5 10 15

<210> 22

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P5

<400> 22

Ser Arg Gln Leu Glu Lys Arg Met Ser Glu Glu Met Arg Leu Leu

1 5 10 15

<210> 23

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P6

<400> 23

Glu Lys Arg Met Ser Glu Glu Met Arg Leu Leu Glu Met Glu Phe

1 5 10 15

<210> 24

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P7

<400> 24

Ser Glu Glu Met Arg Leu Leu Glu Met Glu Phe Glu Glu Arg Lys

1 5 10 15

<210> 25

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P8

<400> 25

Arg Leu Leu Glu Met Glu Phe Glu Glu Arg Lys Arg Ala Ala Glu

1 5 10 15

<210> 26

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P9

<400> 26

Met Glu Phe Glu Glu Arg Lys Arg Ala Ala Glu Gln Arg Gln Lys

1 5 10 15

<210> 27

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P10

<400> 27

Glu Arg Lys Arg Ala Ala Glu Gln Arg Gln Lys Ala Glu Asn Phe

1 5 10 15

<210> 28

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P11

<400> 28

Ala Ala Glu Gln Arg Gln Lys Ala Glu Asn Phe Trp Thr Gly Asp

1 5 10 15

<210> 29

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P12

<400> 29

Arg Gln Lys Ala Glu Asn Phe Trp Thr Gly Asp Thr Ser Ser Ser Asp

1 5 10 15

<210> 30

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P13

<400> 30

Glu Asn Phe Trp Thr Gly Asp Thr Ser Ser Ser Asp Gln Leu Val Leu

1 5 10 15

<210> 31

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P14

<400> 31

Thr Gly Asp Thr Ser Ser Ser Asp Gln Leu Val Leu Gly Lys Lys Asp

1 5 10 15

<210> 32

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P15

<400> 32

Ser Ser Asp Gln Leu Val Leu Gly Lys Lys Asp Met Gly Trp Pro

1 5 10 15

<210> 33

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P16

<400> 33

Leu Val Leu Gly Lys Lys Asp Met Gly Trp Pro Phe Gln Ala Asp

1 5 10 15

<210> 34

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide P17

<400> 34

Lys Lys Asp Met Gly Trp Pro Phe Gln Ala Asp Gly Gln Glu Gly

1 5 10 15

Claims (24)

1. An epitope peptide of ITPRIPL1, the amino acid sequence of the epitope peptide is shown as SEQ ID NO. 25.

2. Use of the epitope peptide of claim 1 for screening for binding proteins capable of efficiently blocking binding of ITPRIPL1 to CD3E and/or SEMA3G, said use comprising contacting a candidate binding protein with said epitope peptide, said candidate binding protein capable of specifically binding to said epitope peptide being identified as capable of efficiently blocking binding of ITPRIPL1 to CD3E and/or SEMA 3G.

3. Use of the epitope peptide of claim 1 for screening for a binding protein capable of promoting killing of tumor cells by PBMC cells, said use comprising contacting a candidate binding protein with said epitope peptide, said candidate binding protein capable of specifically binding to said epitope peptide being identified as capable of promoting killing of tumor cells by PBMC cells.

4. An antibody or antigen binding fragment thereof specifically binding to ITPRIPL1, wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO.7, the amino acid sequence of HCDR2 is shown as SEQ ID NO.8, the amino acid sequence of HCDR3 is shown as SEQ ID NO.9, and wherein the amino acid sequence of LCDR1 is shown as SEQ ID NO.10, the amino acid sequence of LCDR2 is KV, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 11.

5. The antibody or antigen-binding fragment thereof of claim 4, which comprises the heavy chain variable region of amino acid sequence SEQ ID NO.5 and the light chain variable region of amino acid sequence SEQ ID NO. 6.

6. An antibody or antigen-binding fragment thereof specifically binding to ITPRIPL1, wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO.7, the amino acid sequence of HCDR2 is shown as SEQ ID NO.8, and the amino acid sequence of HCDR3 is shown as SEQ ID NO.9, and wherein the amino acid sequence of LCDR1 is shown as SEQ ID NO.16, the amino acid sequence of LCDR2 is KV, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 17.

7. The antibody or antigen-binding fragment thereof of claim 6, comprising the heavy chain variable region of amino acid sequence SEQ ID NO.14 and the light chain variable region of amino acid sequence SEQ ID NO. 15.

8. The antibody or antigen-binding fragment thereof of any one of claims 4-7, comprising an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

9. The antibody or antigen-binding fragment thereof of any one of claims 4-7, comprising an antibody light chain constant region, and the antibody light chain constant region comprises a human Ig kappa constant region.

10. The antibody or antigen-binding fragment thereof of any one of claims 4-5, comprising an antibody heavy chain HC, and the HC comprises the amino acid sequence set forth in SEQ ID NO 3.

11. The antibody or antigen-binding fragment thereof of any one of claims 6-7, comprising an antibody heavy chain HC, and the HC comprises the amino acid sequence set forth in SEQ ID NO 12.

12. The antibody or antigen-binding fragment thereof of any one of claims 4-5, comprising antibody light chain LC, and the LC comprises the amino acid sequence set forth in SEQ ID No. 4.

13. The antibody or antigen-binding fragment thereof of any one of claims 6-7, comprising antibody light chain LC, and the LC comprises the amino acid sequence set forth in SEQ ID NO 13.

14. The antibody or antigen-binding fragment thereof of any one of claims 4-7, wherein the antigen-binding fragment is selected from the group consisting of: fab, fab', F (ab) ₂ Fv fragment, F (ab') ₂ scFv and di-scFv.

15. The antibody or antigen-binding fragment thereof of any one of claims 4-7, wherein the ITPRIPL1 comprises human ITPRIPL1.

16. A chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof of any one of claims 4-15.

17. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 4-15.

18. An isolated one or more nucleic acid molecules encoding the antibody or antigen-binding fragment thereof of any one of claims 4-15 or the chimeric antigen receptor of claim 16.

19. A vector comprising the nucleic acid molecule of claim 18.

20. A cell comprising the nucleic acid molecule of claim 18 or the vector of claim 19.

21. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 4-15, the chimeric antigen receptor of claim 16, the immunoconjugate of claim 17, and optionally a pharmaceutically acceptable adjuvant.

22. A method of making the antibody or antigen-binding fragment thereof of any one of claims 4-15, the method comprising culturing the cell of claim 20 under conditions such that the antibody or antigen-binding fragment thereof of any one of claims 4-15 is expressed.

23. Use of the antibody or antigen-binding fragment thereof of any one of claims 4-15, the chimeric antigen receptor of claim 16, the immunoconjugate of claim 17, and/or the pharmaceutical composition of claim 21 in the manufacture of a medicament for the prevention, amelioration, and/or treatment of a tumor.

24. The use of claim 23, wherein the tumor comprises a lymphoma.

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