Antibody targeting IL1RAP, ADC drug and application of antibody and ADC drug

Abstract

The invention discloses an IL1RAP targeting antibody, an ADC drug and application thereof, and belongs to the technical field of antibody treatment of malignant tumors. According to the invention, the anti-IL1RAP antibody 2D1 is prepared and screened, the anti-IL1RAP antibody 2D1 has high affinity to an IL1RAP antigen, and the KD is 0.005 nM. Furthermore, the antibody drug conjugate (ADC) prepared according to the antibody connected cytotoxic drug has a strong killing effect on IL1RAP positive tumor cells, and the IC50 value is nM level. In-vivo pharmacodynamic experiments prove that the ADC drug has no obvious toxic and side effects on normal tissues and organs while killing tumor cells, and has potential clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of antibody therapy for malignant tumors, specifically relating to an antibody or antigen-binding fragment targeting IL1RAP, an ADC drug containing said antibody or antigen-binding fragment, and their applications. Background Technology

[0002] Liver cancer is a highly malignant tumor. According to the latest China Cancer Report, liver cancer is the fourth most common cancer and the second leading cause of cancer death in my country. It is also the sixth most common cancer and the fourth leading cause of cancer-related death worldwide. It is estimated that by 2025, my country will account for about half of the world's liver cancer patients, with 90% of these cases being hepatocellular carcinoma (HCC). Current treatment methods for liver cancer are still unsatisfactory. Although early-stage HCC has a better prognosis, with a five-year survival rate of 80% or higher, the insidious onset means that 70% of patients are diagnosed at an advanced stage, resulting in a poor prognosis and an overall five-year survival rate of only 11.7%-14.1%. Immunotherapy drugs targeting immune checkpoint inhibitors have recently shown remarkable efficacy in the treatment of advanced liver cancer, but the overall patient response rate is only about 20%, and clinical drug resistance is a serious problem. Therefore, innovative treatments are urgently needed to improve the treatment outcomes of liver cancer and enhance patient prognosis.

[0003] Antibody-drug conjugates (ADCs) are a class of targeted biological agents composed of antibodies, linkers, and cytotoxic drugs. They have demonstrated potent anti-tumor effects and promising therapeutic prospects in numerous cancer types. Monoclonal antibodies possess high specificity, recognizing and binding to specific antigens on the surface of tumor cells, thereby delivering cytotoxic drugs directly to cancer cells, achieving precision treatment, reducing damage to normal cells, and improving treatment efficacy. Furthermore, ADC drugs can overcome the drug resistance problem of traditional chemotherapy, providing new treatment options for cancer patients.

[0004] Currently, there are no clinically available ADC therapies for liver cancer treatment. However, ADC therapies targeting GPC3 are under investigation and have shown promising anti-tumor effects. However, malignant tumors exhibit significant heterogeneity, and a major challenge limiting the further application of ADC therapies is the lack of ideal tumor-specific therapeutic targets. Existing targets such as GPC3, while showing good tumor specificity, still suffer from problems such as poor internalization rates and easy antigen shedding. Other targets, due to their high expression in healthy tissues and poor tumor specificity, pose potential safety concerns and are difficult to apply clinically.

[0005] IL1RAP is an innovative tumor therapeutic target that is highly expressed in hepatocellular carcinoma and many other malignant tumors. This invention develops a high-affinity antibody targeting IL1RAP and a corresponding ADC drug, providing a new potential treatment method for IL1RAP-positive malignant tumors. Summary of the Invention

[0006] One objective of this invention is to obtain a high-affinity monoclonal antibody against IL1RAP, which will serve as a candidate antibody for the subsequent preparation of antibody-drug conjugates. A second objective is to provide a nucleic acid encoding the antibody, an expression vector, and a host cell. A third objective is to provide an antibody-drug conjugate obtained by linking an anti-IL1RAP antibody to a drug, wherein the antibody-drug conjugate exhibits significant in vitro growth inhibition of liver cancer cells and significant in vivo antitumor activity.

[0007] This invention includes the following technical solutions:

[0008] In a first aspect, the present invention discloses an antibody or its antigen-binding fragment having binding specificity for human IL1RAP, characterized in that the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR3 domain of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with ARAGGYSNYYFDY (SEQ ID NO:3); and the light chain variable region comprises a CDR3 domain of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with QQHYSSPWT (SEQ ID NO:7).

[0009] The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the following CDR domains:

[0010] a1) A CDR1 domain of GYTFISYN (SEQ ID NO:1) or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it;

[0011] a2) The CDR2 domain of FYLGNGDT (SEQ ID NO:2) or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and

[0012] a3)ARAGGYSNYYFDY (SEQ ID NO:3) or the CDR3 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it.

[0013] The light chain variable region includes the following CDR structural domains:

[0014] b1) A CDR1 domain of QSVNNE (SEQ ID NO:5) or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it;

[0015] b2) The CDR2 domain of YAS (SEQ ID NO:6) or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and

[0016] b3) QQHYSSPWT (SEQ ID NO:7) or the CDR3 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to it.

[0017] In a preferred embodiment of the present invention, the heavy chain variable region comprises the CDR1 domain of the amino acid sequence shown in SEQ ID NO:1, the CDR2 domain of the amino acid sequence shown in SEQ ID NO:2, and the CDR3 domain of the amino acid sequence shown in SEQ ID NO:3.

[0018] In a preferred embodiment of the present invention, the light chain variable region comprises the CDR1 domain of the amino acid sequence shown in SEQ ID NO:5, the CDR2 domain of the amino acid sequence shown in SEQ ID NO:6, and the CDR3 domain of the amino acid sequence shown in SEQ ID NO:7.

[0019] The antibody or its antigen-binding fragment comprises a heavy chain variable region of SEQ ID NO:4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and a light chain variable region of SEQ ID NO:8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0020] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:4 and the light chain variable region of the amino acid sequence shown in SEQ ID NO:8.

[0021] Furthermore, the antibody or antigen-binding fragment also includes a heavy chain constant region or a portion thereof, the heavy chain constant region being selected from the heavy chain constant regions of the following group of immunoglobulin subtypes: IgG1, IgG2, IgG3 and IgG4.

[0022] In a specific embodiment of the present invention, the heavy chain constant region comprises an amino acid sequence of SEQ ID NO:9 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0023] The antibody or antigen-binding fragment further comprises a light chain constant region or a portion thereof, the light chain constant region being selected from the constant regions of the κ or λ light chain.

[0024] In a specific embodiment of the present invention, the light chain constant region comprises an amino acid sequence of SEQ ID NO:10 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0025] The antibody or antigen-binding fragment comprises a heavy chain of SEQ ID NO:11 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and a light chain of SEQ ID NO:12 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0026] Furthermore, the antibody or its antigen-binding fragment described in this invention also comprises a cytotoxic drug, wherein at least one cytotoxic drug is conjugated to the antibody or its antigen-binding fragment.

[0027] The cytotoxic drugs are selected from one or more of the following: DNA damaging agents, anti-apoptotic agents, mitosis inhibitors, antitumor antibiotics, immunomodulators, nucleic acids for gene therapy, anti-angiogenic agents, antimetabolites, boron-containing agents, hormones, anti-hormonal agents, corticosteroids, photoactive therapeutic agents, radionuclides, radiosensitizers, topoisomerase inhibitors, and tyrosine kinase inhibitors.

[0028] In a second aspect, the present invention discloses a nucleic acid molecule, characterized in that the nucleic acid molecule encodes an antibody or an antigen-binding fragment thereof as described in any of the first aspects of the present invention.

[0029] In one specific embodiment of the present invention, the molecule is a cDNA molecule.

[0030] Thirdly, the present invention discloses a carrier, characterized in that the carrier comprises the nucleic acid molecule described in Part II of the present invention.

[0031] Preferably, the carrier is an expression carrier.

[0032] Fourthly, the present invention discloses a recombinant host cell, characterized in that the recombinant host cell comprises the nucleic acid molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention.

[0033] Preferably, the cells are mammalian host cells. In specific embodiments of the present invention, the host cells include, but are not limited to, Chinese hamster ovary (CHO) cells, NSO melanoma cells, COS cells, and SP2 cells.

[0034] Fifthly, the present invention discloses a method for generating antibodies or antigen-binding fragments, characterized in that the method comprises culturing the recombinant host cells described in the fourth aspect of the present invention under conditions that allow expression of the encoded antibody or its antigen-binding fragment.

[0035] Specifically, the method includes introducing an expression vector encoding an antibody or its antigen-binding fragment into mammalian host cells, wherein the host cells are cultured for a sufficient time to allow the antibody or its antigen-binding fragment to be expressed in the host cells. More preferably, the antibody is secreted into a culture medium in which the host cells are grown, and the antibody or its antigen-binding fragment is isolated using standard protein purification methods.

[0036] In a sixth aspect, the present invention discloses an antibody-drug conjugate (ADC), characterized in that the antibody-drug conjugate comprises an antibody or antigen-binding fragment, and at least one cytotoxic drug conjugated to the antibody or antigen-binding fragment. The definition of the cytotoxic drug herein is the same as that described in the first aspect of the present invention.

[0037] The antibody or antigen-binding fragment provides the ADC with the ability to bind to IL1RAP, enabling the delivery of cytotoxic drugs conjugated / conjugated to the antibody to cells expressing IL1RAP, particularly cancer cells expressing IL1RAP, thereby improving the effectiveness of disease (such as cancer) treatment.

[0038] Furthermore, the cytotoxic drug is conjugated to the antibody or antigen-binding fragment via a connector.

[0039] The connector is a connector commonly used by those skilled in the art, including pyrolytic connectors or non-pyrolytic connectors.

[0040] In a seventh aspect, the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the antibody or antigen-binding fragment thereof described in the first aspect of the present invention and / or the antibody-drug conjugate (ADC) described in the sixth aspect of the present invention.

[0041] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0042] Eighthly, the present invention discloses the use of any of the following in the preparation of a medicament for treating diseases related to IL1RAP:

[0043] 1) The antibody or antigen-binding fragment thereof as described in the first aspect of this invention;

[0044] 2) The nucleic acid molecule described in the second aspect of this invention;

[0045] 3) The expression vector described in the third aspect of this invention;

[0046] 4) The recombinant host cell described in the fourth aspect of this invention;

[0047] 5) The antibody-drug conjugate (ADC) as described in the sixth aspect of this invention;

[0048] 6) The pharmaceutical composition according to the seventh aspect of the present invention.

[0049] The diseases associated with IL1RAP are those known to those skilled in the art that are positive for IL1RAP expression, including hematologic malignancies and solid tumors.

[0050] The hematologic malignancies mentioned include, but are not limited to, acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndrome.

[0051] The solid tumors include, but are not limited to, liver cancer, prostate cancer, breast cancer, lung cancer, colon cancer, melanoma, bladder cancer, brain cancer, cervical cancer, pharyngeal cancer, stomach cancer, head and neck cancer, kidney cancer, ovarian cancer, lymphoma, pancreatic cancer, and sarcoma.

[0052] In a specific embodiment of the present invention, the disease associated with IL1RAP is hepatocellular carcinoma.

[0053] In specific embodiments of the present invention, the use includes administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof described in the present invention, or the antibody-drug conjugate (ADC) or pharmaceutical composition described in the present invention to a subject in need.

[0054] The treatment also includes administering the antibody or its antigen-binding fragment described in this invention, or the antibody-drug conjugate (ADC) described in this invention, or the pharmaceutical composition in combination with other agents or other treatment methods.

[0055] Specifically, the other agents include, but are not limited to, immune checkpoint inhibitors and chemotherapy agents. The immune checkpoint inhibitors include antibodies, such as anti-PD1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies.

[0056] Other treatment methods include, but are not limited to, radiotherapy.

[0057] The anti-IL1RAP antibody or its antigen-binding fragment prepared by this invention has high affinity for the IL1RAP antigen. D The antibody-drug conjugate (ADC) prepared according to the above method, with a concentration of 0.005 nM, exhibits potent cytotoxic activity against IL1RAP-positive tumor cells, with an IC50 concentration of 0.005 nM. 50 The value is in the nM range, and the ADC drug has no obvious toxic side effects on normal tissues and organs while killing tumor cells, thus having potential clinical application value.

[0058] The amino acid and nucleotide sequences involved in this invention are as follows:

[0059]

[0060]

[0061] Attached Figure Description

[0062] Figure 1 BLI assays the affinity of the monoclonal antibody 2D1 targeting IL1RAP for the antigen.

[0063] Figure 2 In vitro killing effect of ADC drugs targeting IL1RAP on Huh-7 cell line.

[0064] Figure 3 In vitro killing effect of ADC drugs targeting IL1RAP on Hep3B cell line.

[0065] Figure 4 In vitro killing effect of ADC drugs targeting IL1RAP on JHH7 cell line.

[0066] Figure 5 In vivo antitumor activity of ADC drugs targeting IL1RAP conjugated with MMAF.

[0067] Figure 6In vivo antitumor activity of ADC drugs targeting IL1RAP conjugated to SN38.

[0068] Figure 7 Safety evaluation of ADC drugs targeting IL1RAP conjugated with MMAF.

[0069] Figure 8 Safety evaluation of ADC drugs targeting IL1RAP conjugated to SN38. Detailed Implementation

[0070] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0071] Terminology Explanation

[0072] The term "CDR" refers to the complementarity-determining region within the variable sequence of an antibody. For each variable region, there are three CDRs in each variable region of the heavy chain (HC) and the light chain (LC), named CDR1, CDR2, and CDR3, respectively.

[0073] The term "amino acid sequence identity percentage (%)" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, after alignment and, if necessary, introducing gaps to achieve the maximum sequence identity percentage, without considering any conserved substitutions as part of sequence identity. For the purpose of determining the amino acid sequence identity percentage, alignment can be performed in various ways within the scope of the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0074] Term "K" D "Refers to the equilibrium dissociation constant of a specific antibody-antigen interaction."

[0075] The term "IL1RAP-associated disease" includes any condition or disease (including proliferative conditions such as cancer) that is marked, diagnosed, detected, or identified by phenotypic or genotypic abnormalities of IL1RAP genetic components or expression during the course or etiology of the disease or condition. In this regard, IL1RAP phenotypic abnormalities or determinants may, for example, include increased or decreased IL1RAP protein expression levels in a cell population (e.g., a cancer cell population) compared to another cell population (e.g., a normal cell population), or increased or decreased IL1RAP protein expression in certain definable cell populations, or increased or decreased IL1RAP protein expression at inappropriate times or stages of the cell life cycle. Furthermore, similar expression patterns of IL1RAP genotypic determinants (e.g., mRNA transcriptional levels) may also be used to classify or detect IL1RAP-associated conditions. In one embodiment, an IL1RAP-associated disease is hepatocellular carcinoma.

[0076] The term "combination therapy" refers to the administration of another therapeutic agent concurrently with, before, or after the administration of an anti-IL1RAP antibody or ADC. In one embodiment, the anti-IL1RAP antibody or ADC of the present invention is administered in combination with one or more immune checkpoint inhibitors used to treat cancer.

[0077] The term "effective dose" refers to a dose sufficient to demonstrate its benefit to the recipient. The actual amount administered, as well as the rate and duration of administration, will depend on the individual's condition and the severity of the illness.

[0078] The term "pharmaceutical composition" refers to a formulation in which one or more active ingredients are biologically effective and free from other components that would have unacceptable toxicity to a subject to which the formulation is administered. Therefore, it is a composition suitable for pharmaceutical use in mammalian subjects (typically humans). Pharmaceutical compositions typically contain effective amounts of an active agent and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively. Such formulations may be sterile.

[0079] The term "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulation material, formulation aid, or carrier conventional in the art, used in conjunction with a therapeutic agent to constitute a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used and is compatible with the other components of the formulation. A pharmaceutically acceptable carrier is suitable for use in the formulation.

[0080] The term "treatment" refers to a clinical intervention that attempts to alter the natural course of the disease in the individual being treated, whether for prevention or in the course of clinicopathology. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, relieving symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and eliminating or improving prognosis.

[0081] Example 1: Preparation of IL1RAP monoclonal antibody

[0082] 1.1 Preparation of antigen

[0083] The mRNA and amino acid sequences of IL1RAP were retrieved from the NCBI database. The extracellular segment of IL1RAP (Ser21-Lys350) was fused with Human IgG1 FC and cloned into pcDNA3.1 to construct pcDNA3.1-IL1RAP-FC. In addition, an HIS tag was added to the C-terminus of the extracellular segment of IL1RAP (Ser21-Lys350) to construct pcDNA3.1-IL1RAP-HIS vector, which was then transformed into TOP10 competent cells.

[0084] The above-mentioned strains were cultured, bacterial cells were collected, and plasmids were extracted using a plasmid extraction kit (Tiangen Biotech Co., Ltd.). The plasmids were then transiently transfected into HEK293F cells. Seven days after transfection, the cell culture supernatant was collected, and IL1RAP-FC protein was purified using Protein A packing material. IL1RAP-HIS protein was then purified using a NI column.

[0085] 1.2 Immunization of mice

[0086] Six 6-week-old Balb / c mice were selected. For the first immunization, 100 μg of IL1RAP-FC protein was mixed with PAP-1 adjuvant (Siger Biotech, catalog number: PAP-1) according to the product instructions and injected intraperitoneally into each mouse. For the second and third immunizations, the antigen dosage was 50 μg, and the adjuvant and immunization method remained the same. Three days before hybridoma fusion, the mice were immunized intraperitoneally with 50 μg of IL1RAP-HIS protein.

[0087] 1.3 Preparation of hybridoma cells

[0088] Mouse spleen cells were isolated and fused with mouse myeloma cells by electrofusion. The resulting hybridoma cells were used to screen for anti-IL1RAP specific antibodies. After electrofusion, the cells were allowed to stand for 10 minutes and then transferred to screening medium. The cells were incubated at 37°C for 1 hour, plated, and the medium was changed every 2 days. The cells were tested after 10 days.

[0089] 1.4 Subcloning and Screening

[0090] Indirect ELISA and flow cytometry were used to screen culture wells with high titers for subcloning after staining positive tumor cell lines in myeloma cell supernatant. Subcloning was repeated until the positive rate of cell lines in the wells reached 100%, and the positive hybridoma cells were expanded for culture.

[0091] 1.5 Monoclonal Antibody Acquisition and Purification

[0092] A single-clonal cell line was obtained, and the selected clone was named 2D1 and sequenced. A chimeric antibody was constructed by combining the variable region of the selected clone with the constant region of human IgG1, transiently transexpressed using HEK293F, purified in one step using protein A, and its affinity was determined. Results are as follows: Figure 1 As shown, the K of antibody 2D1 D The value is 0.005nM.

[0093] Example 2: Preparation of ADC drugs

[0094] Prepare a 10×TECP solution by mixing TECP and antibody at a ratio of 10:1 and reducing the antibody at 25°C and 100 rpm for 30 min. Prepare a 10×DTPA solution by adding DTPA to a final concentration of 1 mM and reducing at 25°C and 100 rpm for 1-1.5 h. Prepare a 10 mM drug solution using DMSO and add it to the reaction system at a ratio of 5:1 to the antibody, and couple at 25°C and 100 rpm for 1-1.5 h. Terminate the reaction by adding 10-fold N-acetylcysteine. Dialyze using PBS with slow stirring at 4°C for 8-16 h. The final dilution factor is ≥40000, and the solution is concentrated to 500 μl using an ultrafiltration tube.

[0095] The present invention prepares the following ADC drugs according to the above method: 2D1-SN38, 2D1-MMAF, Isotype-SN38, and Isotype-MMAF.

[0096] SN38 is the active metabolite of irinotecan, a topoisomerase I inhibitor, with the following structure:

[0097]

[0098] MMAF, or monomethylauratestatin F, is an anti-microtubule inhibitor.

[0099] CL2A is a degradable PBC-peptide-mc linker containing PEG8 and triazo. CL2A undergoes pH-sensitive degradation, produces a bystander effect, and binds to antibodies on cysteine ​​residues via disulfide bonds.

[0100] vcMMAF is a conjugate drug with antitumor activity, consisting of the anti-microtubule inhibitor MMAF and the cathepsin-degradable linker MC-Val-Cit-PAB.

[0101] Isotype is a human IgG1 control antibody that has no significant affinity for the target antigen.

[0102] Example 1: In vitro efficacy experiment

[0103] In vitro pharmacological experiments were conducted using Huh-7, Hep3B, and JHH7 cells as target cells. Cell viability and cell count were determined using the CCK8 assay, and the cell density was adjusted to 1×10⁶ cells / year. 5 100 μl of diluted test sample was added to each well of a 96-well black flat-bottom cell culture plate. 20 μl of the diluted test sample was added to each well of the pre-seeded 96-well cell culture plate. The plates were incubated in a cell culture incubator (37℃, 5% CO2) for 72±3 h. CCK8 reagent was then added, and the readings were taken at 450 nm using a microplate reader. The IC50 of the ADC drug was obtained by fitting the detection results. 50 value.

[0104] Drugs for in vitro cell experiments: 2D1, 2D1-SN38, 2D1-MMAF, CL2A-SN38, Isotype-SN38, vcMMAF, Isotype-MMAF.

[0105] Test results as follows Figure 2-4 As shown, in in vitro cell experiments using Huh-7, Hep3B, and JHH7 cells as target cells, compared to 2D1, the groups treated with ADC drugs (such as 2D1-SN38, 2D1-MMAF, CL2A-SN38, Isotype-SN38, vcMMAF, and Isotype-MMAF) had lower cell survival rates and better tumor cell killing effects. A horizontal comparison of 2D1-SN38, CL2A-SN38, and Isotype-SN38, with the same conjugated drugs, showed that 2D1-SN38 prepared in this invention exhibited better tumor cell killing activity. Similarly, comparing 2D1-MMAF, vcMMAF, and Isotype-MMAF, with the same conjugated drugs, 2D1-MMAF prepared in this invention showed better tumor cell killing effects. These results demonstrate that the anti-IL1RAP antibody 2D1 prepared and screened in this invention has better specificity against liver cancer cells, and its IC50 value is significantly higher than that of 2D1-SN38. 50 The value is in the nM range.

[0106] Example 2: In vivo efficacy experiment

[0107] Establishment of tumor-bearing mouse model: Fifteen BALB / c nude mice were randomly divided into three groups of five each. Each mouse was subcutaneously injected with 5 × 10⁻⁶ mol / L tumor-bearing mice. 6 One Huh-7 cell. When the tumor volume reaches 50-100 mm. 3 Mice were administered the drug via tail vein at a dose of 5 mg / kg, once every 3 days for a total of 4 administrations. Tumor volume and mouse weight were measured on days 1, 4, 7, 10, 13, 16, 19, and 22 after administration. A blank control group was administered the same dose of PBS.

[0108] The grouping and drug administration details for the in vivo efficacy experiment are as follows:

[0109] Experiment 1: 2D1-MMAF group, Isotype-MMAF group (represented by ISO-MMAF in the figure), PBS group.

[0110] Experiment 2: 2D1-SN38 group, Isotype-SN38 group (represented as ISO-SN38 in the figure), PBS group.

[0111] The results are as follows Figure 5-8 As shown, compared with the ISO-MMAF and ISO-SN38 administration groups, the tumor volume in the 2D1-MMAF and 2D1-SN38 administration groups decreased more significantly after administration, indicating that the ADC drug prepared in this invention has a better tumor-killing effect, and the anti-IL1RAP antibody 2D1 prepared in this invention has a better targeted delivery effect on liver cancer cells. Figure 7 and Figure 8 As can be seen, the ADC drugs 2D1-MMAF and 2D1-SN38 prepared in this invention, which have better tumor killing effects, did not have a significant effect on the body weight of mice, suggesting that they have good safety.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody or its antigen-binding fragment, having binding specificity for human IL1RAP, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises a CDR3 domain of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with ARAGGYSNYYFDY (SEQ ID NO:3). The light chain variable region comprises a CDR3 domain of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with QQHYSSPWT (SEQ ID NO:7).

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the following CDR domains: a1) A CDR1 domain of GYTFISYN (SEQ ID NO:1) or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; a2) The CDR2 domain of FYLGNGDT (SEQ ID NO:2) or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and a3) ARAGGYSNYYFDY (SEQ ID NO:3) or the CDR3 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it; The light chain variable region includes the following CDR structural domains: b1) A CDR1 domain of QSVNNE (SEQ ID NO:5) or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it; b2) The CDR2 domain of YAS (SEQ ID NO:6) or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and b3) QQHYSSPWT (SEQ ID NO:7) or the CDR3 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to it.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region of SEQ ID NO:4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and a light chain variable region of SEQ ID NO:8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes an antibody or antigen-binding fragment of any one of claims 1-3.

5. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 4.

6. Recombinant host cells, characterized in that, The recombinant host cell comprises the nucleic acid molecule of claim 4 or the vector of claim 5.

7. A method for generating the antibody or antigen-binding fragment according to any one of claims 1-3, characterized in that, The method includes culturing the recombinant host cell of claim 6 under conditions that allow expression of the encoded antibody or its antigen-binding fragment.

8. An antibody-drug conjugate (ADC), characterized in that, The antibody-drug conjugate / conjugate comprises the antibody or antigen-binding fragment as described in any one of claims 1-3, and at least one cytotoxic drug conjugated to the antibody or antigen-binding fragment.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-3 and / or the antibody-drug conjugate (ADC) as described in claim 8.

10. The use of any of the following in the preparation of a medicament for treating diseases related to IL1RAP: 1) The antibody or antigen-binding fragment thereof as described in any one of claims 1-3; 2) The nucleic acid molecule as described in claim 4; 3) The expression vector as described in claim 5; 4) The recombinant host cell as described in claim 6; 5) The antibody-drug conjugate (ADC) as described in claim 8; 6) The pharmaceutical composition according to claim 9.

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