Application of medicament for inhibiting ICAM-1-FGG interaction in preparation of medicament for treating cancer

Abstract

The invention discloses an application of a medicament for inhibiting the interaction of ICAM-1-FGG in preparation of medicaments for treating cancers. Also disclosed is a method of apoptosis of cancer cells comprising treating cancer cells and inducing apoptosis thereof with a blocker that inhibits the ICAM-1-FGG interaction. The invention further discloses an antibody for resisting ICAM-1 and application of the antibody, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3. The invention discloses a new mechanism that ICAM-1 provides a survival signal for cancer, and the ICAM-1 can be used as a potential molecular target for cancer treatment by blocking the interaction of ICAM-1-FGG. The anti-ICAM-1 antibody provided by the invention can effectively cause apoptosis of cancer cells and inhibit in-vivo growth of the cancer cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of an agent for inhibiting ICAM-1-FGG interaction in preparing a drug for treating cancer. Background Art

[0002] Lung cancer is one of the cancers with the highest mortality rate in the world. Therefore, studying the mechanism of lung cancer occurrence and development is of great significance for the development and treatment of lung cancer drugs. An important characteristic of malignant tumors is that cells lack normal apoptosis mechanisms and can proliferate indefinitely, which is also an important reason for the patient's eventual imbalance and death. In addition to genetic changes inside the tumor, receptors on the surface of tumor cells may participate in the regulation of tumor cell apoptosis signals by receiving signals from the environment. Among the many receptor molecules on the surface of tumor cells, cell adhesion molecules (CAM) are a type of bridge molecules that connect the inside and outside of cells. Cell adhesion molecules are essential in many important life activities such as cell proliferation, survival and migration. At the same time, cell adhesion molecules are also involved in regulating the occurrence, infiltration and metastasis of tumors, which is a hot and cutting-edge topic in tumor research. In malignant tumors, the abnormal expression of cell adhesion molecules not only affects cell adhesion, but also affects cell survival and other functions.

[0003] Intercellular cell adhesion molecule-1 (ICAM-1), also known as CD54, belongs to the immunoglobulin superfamily and is a single-pass transmembrane glycoprotein molecule. Its molecular weight ranges from 74 to 114 KDa due to different degrees of glycosylation. ICAM-1 consists of an extracellular region including 5 immunoglobulin-like domains, a transmembrane region, and a short intracytoplasmic tail. The extracellular head can bind to a variety of molecules, and the intracellular region also contains skeletal protein and signal molecule binding sites. There is increasing evidence that the expression of ICAM-1 is also upregulated during tumorigenesis, and its relationship with tumors has also begun to attract the attention of many scholars. Studies have found that the expression of ICAM-1 affects the proliferation of tumor cells. Knocking down the expression of ICAM-1 in tumor cells inhibits the proliferation of cells, while overexpression of ICAM-1 promotes tumor growth.

[0004] Recent studies have found that under the stimulation of inflammatory factors, epithelial cells will overexpress ICAM-1, which promotes the survival of epithelial cells under inflammatory conditions by binding to the surface receptor fibrinogen (FG). FG is a dimer composed of three chains in pairs, α, β, and γ, with a molecular weight of about 340KDa. The genes encoding the three polypeptide chains are FGA, FGB, and FGG. Among them, the FGγ chain (FGG) (117-133) site can directly bind to the ICAM-1 (8-22) amino acid sequence, thereby participating in the regulation of cell-to-cell interactions and signal transmission. Summary of the invention

[0005] In order to solve the defect that the role of ICAM-1-FGG interaction in the development of cancer is still unknown in the prior art, the present invention provides the use of an agent that inhibits ICAM-1-FGG interaction in the preparation of a drug for treating cancer. The present invention reveals a new mechanism by which ICAM-1 provides survival signals for a variety of solid tumor cells including non-small cell lung cancer, and blocking ICAM-1-FGG interaction can be used as a potential molecular target for cancer treatment.

[0006] The present invention mainly finds that ICAM-1 maintains the survival signal of non-small cell lung cancer by binding to FGG secreted by tumors. Under normal circumstances, ICAM-1 expressed by non-small cell lung cancer cells binds to the FGG 117-133 sequence secreted by them, regulates the phosphorylation of the intracellular segment of ICAM-1 and recruits the phosphatase SHP-2 to bind to the tail domain of ICAM-1, thereby maintaining the activation of cell survival-related kinases such as ERK and Akt, while inhibiting the phosphorylation of JNK and p 38MAPK, and ultimately maintaining the survival and proliferation signals of cancer cells. Once the interaction between ICAM-1 and FGG is destroyed, the intracellular segment of ICAM-1 will undergo dephosphorylation and lose the binding of SHP-2, the activation of key cell survival kinases ERK and Akt is inhibited, the activation of stress and apoptosis-related kinases such as JNK and p38MAPK is significantly upregulated, and non-small cell lung cancer cells initiate apoptosis programs dependent on the caspase-9 / 3 signaling pathway, ultimately affecting the survival of tumor cells in vitro and the progression in vivo. Therefore, it is believed that blocking the interaction between ICAM-1 and FGG is one of the effective ways to induce tumor apoptosis and control tumor growth.

[0007] To solve the above technical problems, one of the technical solutions provided by the present invention is: an anti-ICAM-1 antibody, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3; wherein: the amino acid sequence of the LCDR1 is as shown in SEQ ID NO:6, the amino acid sequence of the LCDR2 is RAS, the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:7, the amino acid sequence of the HCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:9, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:10.

[0008] In a specific embodiment of the present invention, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:11; and / or, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:12.

[0009] In a specific embodiment of the present invention, the antibody is a full-length antibody, Fab, Fab', F(ab')2 or Fv.

[0010] In a preferred embodiment of the present invention, the Fv is a scFv.

[0011] In a specific embodiment of the present invention, the antibody is a full-length antibody, and the heavy chain constant region and / or the light chain constant region thereof are derived from a human antibody.

[0012] In a preferred embodiment of the present invention, the heavy chain constant region and / or the light chain constant region are derived from human IgG1 antibody.

[0013] In a more preferred embodiment of the present invention, the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO:13; and / or, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO:14.

[0014] To solve the above technical problems, the second technical solution provided by the present invention is: an isolated nucleic acid, wherein the nucleic acid encodes the antibody as described in one of the technical solutions of the present invention.

[0015] In order to solve the above technical problems, the third technical solution provided by the present invention is: a recombinant expression vector, which comprises the isolated nucleic acid as described in the second technical solution of the present invention.

[0016] To solve the above technical problems, the fourth technical solution provided by the present invention is: a transformant, which comprises the nucleic acid as described in the second technical solution of the present invention or the recombinant expression vector as described in the third technical solution of the present invention, and the host cell of the transformant is a eukaryotic cell or a prokaryotic cell.

[0017] To solve the above technical problems, the fifth technical solution provided by the present invention is: a method for preparing an anti-ICAM-1 antibody, the method comprising culturing the transformant as described in the fourth technical solution of the present invention.

[0018] To solve the above technical problems, the sixth technical solution provided by the present invention is: a pharmaceutical composition, which comprises the antibody as described in one of the technical solutions of the present invention, and a pharmaceutically acceptable carrier.

[0019] In order to solve the above technical problems, the seventh technical solution provided by the present invention is: a chimeric antigen receptor, wherein the chimeric antigen receptor comprises the antibody as described in one of the technical solutions of the present invention.

[0020] In order to solve the above technical problems, the eighth technical solution provided by the present invention is: a genetically modified cell, wherein the genetically modified cell comprises the chimeric antigen receptor as described in the seventh technical solution of the present invention.

[0021] In a preferred embodiment of the present invention, the genetically modified cells are eukaryotic cells, preferably isolated human cells; more preferably immune cells such as T cells or NK cells.

[0022] To solve the above technical problems, the ninth technical solution provided by the present invention is: an antibody-drug conjugate, which comprises a cytotoxic agent or a label, and the antibody as described in one of the technical solutions of the present invention.

[0023] To solve the above technical problems, the tenth technical solution provided by the present invention is: a kit, which comprises the antibody as described in one of the technical solutions of the present invention, the pharmaceutical composition as described in the sixth technical solution of the present invention, the chimeric antigen receptor as described in the seventh technical solution of the present invention, the genetically modified cell as described in the eighth technical solution of the present invention and / or the antibody-drug conjugate as described in the ninth technical solution of the present invention.

[0024] To solve the above technical problems, the eleventh technical solution provided by the present invention is: a set of medicine kits, comprising medicine kit A and medicine kit B, wherein: the medicine kit A contains the antibody as described in one of the technical solutions of the present invention, the pharmaceutical composition as described in the sixth technical solution of the present invention, the chimeric antigen receptor as described in the seventh technical solution of the present invention, the genetically modified cell as described in the eighth technical solution of the present invention and / or the antibody-drug conjugate as described in the ninth technical solution of the present invention; the medicine kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and / or other anti-tumor drugs.

[0025] To solve the above technical problems, the twelfth technical solution provided by the present invention is: a drug delivery device, which comprises the antibody as described in one of the technical solutions of the present invention, the pharmaceutical composition as described in the sixth technical solution of the present invention, the chimeric antigen receptor as described in the seventh technical solution of the present invention, the genetically modified cell as described in the eighth technical solution of the present invention and / or the antibody-drug conjugate as described in the ninth technical solution of the present invention.

[0026] In a preferred embodiment of the present invention, the drug delivery device further comprises a component for administering the antibody, the chimeric antigen receptor, the antibody drug conjugate, the genetically modified cell or the pharmaceutical composition to a subject, such as a syringe or an infusion device.

[0027] To solve the above technical problems, the thirteenth technical solution provided by the present invention is: a method for immunodetection or determination of a protein comprising an ICAM-1 8-22 antigen polypeptide (SEQ ID NO: 17), comprising using the antibody as described in one of the technical solutions of the present invention, the pharmaceutical composition as described in the sixth technical solution of the present invention, the chimeric antigen receptor as described in the seventh technical solution of the present invention, the genetically modified cell as described in the eighth technical solution of the present invention and / or the antibody-drug conjugate as described in the ninth technical solution of the present invention; the method is for non-diagnostic purposes.

[0028] In a preferred embodiment of the present invention, the protein comprising the ICAM-1 8-22 antigen polypeptide is ICAM-1.

[0029] To solve the above technical problems, the fourteenth technical solution provided by the present invention is: use of the antibody as described in one of the technical solutions of the present invention, the pharmaceutical composition as described in the sixth technical solution of the present invention, the chimeric antigen receptor as described in the seventh technical solution of the present invention, the genetically modified cell as described in the eighth technical solution of the present invention and / or the antibody-drug conjugate as described in the ninth technical solution of the present invention in the preparation of a preparation for inhibiting the binding of a protein comprising an ICAM-1 8-22 antigen polypeptide (SEQ ID NO: 17) and a protein comprising a fibrinogen γ chain (SEQ ID NO: 16).

[0030] In a preferred embodiment of the present invention, the protein comprising the ICAM-1 8-22 antigen polypeptide is ICAM-1 and / or the protein comprising the fibrinogen γ chain is fibrinogen.

[0031] In order to solve the above technical problems, the fifteenth technical solution provided by the present invention is: a method for causing apoptosis of cancer cells, the method comprising treating cancer cells with a blocker that inhibits ICAM-1-FGG interaction and inducing apoptosis of cancer cells, wherein the cancer cells rely on ICAM-1-FGG interaction to maintain survival; the method is for non-therapeutic purposes.

[0032] In a preferred embodiment of the present invention, the cancer cells are lung cancer cells, colorectal cancer cells, pancreatic cancer cells or renal cancer cells; the lung cancer cells are preferably non-small cell lung cancer cells, and the colorectal cancer cells are preferably colon adenocarcinoma cells; and / or the inhibitor of ICAM-1–FGG interaction is an antibody, a polypeptide or a small molecule drug, a chimeric antigen receptor comprising the antibody, an antibody-drug conjugate, a genetically modified cell or a pharmaceutical composition, or a pharmaceutical composition comprising the polypeptide or small molecule drug.

[0033] In a more preferred embodiment of the present invention, the non-small cell lung cancer cells are A549, H1650, H1299 or HCC827, the colon adenocarcinoma cells are SW480, HT-29 or HCT 116, the pancreatic cancer cells are PANC-1, and the renal cancer cells are 769-P or 786-O; and / or, the blocker for inhibiting ICAM-1–FGG interaction is an antibody as described in one of the technical solutions of the present invention, a pharmaceutical composition as described in the sixth technical solution of the present invention, a chimeric antigen receptor as described in the seventh technical solution of the present invention, a genetically modified cell as described in the eighth technical solution of the present invention, or an antibody-drug conjugate as described in the ninth technical solution of the present invention.

[0034] To solve the above technical problems, the sixteenth technical solution provided by the present invention is: use of a blocker that inhibits ICAM-1–FGG interaction in the preparation of a drug for diagnosing, preventing and / or treating cancer; cancer cells in the cancer rely on ICAM-1–FGG interaction to maintain survival.

[0035] In a preferred embodiment of the present invention, the cancer is lung cancer, colorectal cancer, pancreatic cancer or renal cancer; the lung cancer is preferably non-small cell lung cancer, and the colorectal cancer is preferably colon adenocarcinoma; and / or the inhibitor of inhibiting ICAM-1-FGG interaction is an antibody, a polypeptide or a small molecule drug, a chimeric antigen receptor comprising the antibody, an antibody-drug conjugate, a genetically modified cell or a pharmaceutical composition, or a pharmaceutical composition comprising the polypeptide or small molecule drug.

[0036] In a more preferred embodiment of the present invention, the inhibitor of ICAM-1–FGG interaction is an antibody as described in one of the technical solutions of the present invention, a pharmaceutical composition as described in the sixth technical solution of the present invention, a chimeric antigen receptor as described in the seventh technical solution of the present invention, a genetically modified cell as described in the eighth technical solution of the present invention, or an antibody-drug conjugate as described in the ninth technical solution of the present invention.

[0037] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0038] The reagents and raw materials used in the present invention are commercially available.

[0039] The positive and progressive effects of the present invention are:

[0040] The present invention reveals a new mechanism by which ICAM-1 provides survival signals for a variety of solid tumor cells, including non-small cell lung cancer, colorectal cancer, pancreatic cancer and renal cancer, and blocking the ICAM-1–FGG interaction can be used as a potential molecular target for the treatment of a variety of cancers. Using blocking antibodies or peptides to specifically disrupt the interaction between tumor cell ICAM-1 and autocrine FGG may have a certain impact on the survival and progression of ICAM-1-positive cancers. At the same time, disrupting this interaction will not affect the binding of ICAM-1 to its main ligand β2 integrin, thereby avoiding the side effects on the immune system's recognition and tumor killing process.

[0041] The antibody for inhibiting the binding of intercellular adhesion molecule-1 to fibrinogen provided by the present invention has an affinity dissociation constant of 9.15 nM for ICAM-1 protein, and a half-inhibitory concentration of 0.89 μg / ml for inhibiting the binding of FG protein to non-small cell lung cancer cells. It can effectively cause the activation of apoptosis-related caspase-9 / 3 protein and apoptosis of cancer cells, and can effectively inhibit the growth of cancer cells in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1ICAM-1 expression in common malignant tumor types and enrichment analysis results of genes related to non-small cell lung cancer. (A) Box-and-whiskers diagram of ICAM-1 expression in common tumors in the TCGA database (https: / / portal.gdc.cancer.gov / ). The midpoint of the diagram indicates the outlier, the midline indicates the median, and the boundaries indicate the quartiles. (B) ICAM-1 staining levels in sections of non-small cell lung cancer cases in the Human Protein Atlas (HPA) database (http: / / www.proteinatlas.org / ). Left, representative images of strong and low / no signal results of non-small cell lung cancer sections stained with anti-ICAM-1 antibody (HPA004877). Scale bar, 200μm. Right, statistical results of HPA staining data. The level of immunohistochemical staining (staining) is determined by both the staining intensity (intensity) and the staining quantity (quantity), where the staining intensity indicates the depth of antibody staining and the staining quantity indicates the proportion of tumor cells in the section that are positively stained. (C) GSEA gene enrichment analysis results of ICAM-1 expression in non-small cell lung cancer cases. NES, normalized enrichment score; FDR, false discovery rate.

[0043] Figure 2 Expression of ICAM-1 in non-small cell lung cancer cells. Expression of ICAM-1 on the surface of normal lung bronchial epithelial cells 16HBE and different non-small cell lung cancer cell lines. The left figure shows the representative results of three independent repeated experiments. The data show the mean ± standard deviation of three independent repeated experiments (n = 3). ***P < 0.001.

[0044] Figure 3 Detection of cell apoptosis after knockdown of ICAM-1 on the surface of normal lung epithelial cells 16HBE and non-small cell lung cancer cells A549 and H1650. (A) Knockdown efficiency and recovery of ICAM-1 on the surface of normal lung epithelial cells 16HBE and non-small cell lung cancer cells A549 and H1650. The above figure is a representative result of three independent repeated experiments. The data show the mean ± standard deviation of three independent repeated experiments (n = 3). (B) Live cells were labeled with Annexin V-FITC and PI double negative, FITC + / PI - Indicates early apoptotic cells, FITC + / PI +Indicates late apoptotic cells. The above figure shows the representative results of three independent repeated experiments. The data show the mean ± standard deviation of three independent repeated experiments (n = 3). **P < 0.01, ***P < 0.001, NS no significant difference (P > 0.05).

[0045] Figure 4 Expression of ICAM-1 ligand protein in non-small cell lung cancer cells A549 and H1650. (A) Expression of integrin β2 on the surface of normal lung bronchial epithelial cells 16HBE and different non-small cell lung cancer cell lines. Human T lymphocyte line Jurkat was used as a positive control for the expression of integrin β2. The values ​​in the table indicate the mean fluorescence intensity (MFI) of ICAM-1 antibody (HA58) staining. The data show the mean ± standard deviation of three independent repeated experiments (n=3). (B) Immunoblotting assay to detect the expression of MUC1 in 16HBE, A549 and H1650 cells, and human breast cancer cells MCF7 were used as positive controls. The figure shows the representative results of three independent repeated experiments. (C) Immunoblotting assay to detect the expression of fibrinogen γ chain (FGG) in 16HBE, A549 and H1650 cells, and ICAM-1 antibody was used to immunoprecipitate FGG in A549 and H1650 cells. The figure shows the representative results of three independent repeated experiments. (D) Detection of cell apoptosis after FGG knockdown in non-small cell lung cancer cells A549 and H1650. Immunoblotting was used to detect the knockdown efficiency of FGG in cells (left), and flow cytometry was used to detect cell apoptosis (right). The figure shows the representative results of three independent repeated experiments. The data show the mean ± standard deviation of three independent repeated experiments (n = 3). ***P < 0.001.

[0046] Figure 5 The apoptosis of non-small cell lung cancer cells A549 (A) and H1650 (B) after knockdown of ICAM-1 was detected after re-expression of ICAM-1 8-22 deletion mutant and intracellular segment deletion mutant. Live cells were labeled with Annexin V-FITC and PI double negative, FITC + / PI - Indicates early apoptotic cells, FITC + / PI + Indicates late apoptotic cells. The figure above shows the knockdown efficiency and recovery of ICAM-1 on the cell surface. The figure shows the representative results of three independent repeated experiments. The data show the mean ± standard deviation of three independent repeated experiments (n = 3). ***P < 0.001, NS no significant difference (P > 0.05).

[0047] Figure 6Detection of apoptosis signals after ICAM-1 knockdown and reversion in non-small cell lung cancer cells A549 and H1650. (A) Immunoblotting assay was used to detect the expression of apoptosis signaling-related proteins caspase-9 / 3 and caspase-8 in A549 and H1650 cells. (B) Immunoblotting assay was used to detect the tyrosine phosphorylation of the intracellular domain of ICAM-1 and the binding of phosphatase SHP-2 in A549 and H1650 cells. (C) Immunoblotting assay was used to detect the phosphorylation of Akt (Ser473), ERK1 / 2 (Thr202 / Tyr204), JNK (Thr183 / Tyr185) and p38 (Thr180 / Tyr182) in A549 and H1650 cells. The figure shows the representative results of three independent repeated experiments.

[0048] Figure 7 Expression of ICAM-1 and FGG in nude mouse A549 subcutaneous tumors. Immunoblotting assays were performed to detect the expression of ICAM-1 and FGG in A549 subcutaneous tumors treated with control shRNA, ICAM-1 and FGG-specific shRNA, and ICAM-1 knockdown and restoration of ICAM-1Δ8-22 mutant protein and wild-type ICAM-1 protein. Representative results of three independent replicates are shown.

[0049] Figure 8 Disruption of ICAM-1-FGG interaction inhibits the growth of non-small cell lung cancer implants in mice. (A) Growth of A549 subcutaneous tumors in nude mice transfected with control shRNA, ICAM-1 or FGG-specific shRNA, and after knockdown of ICAM-1, the expression of ICAM-1Δ8-22 mutant protein and wild-type ICAM-1 protein was restored. (B) Tumor size of each experimental group 15 days after subcutaneous tumor implantation. (C) TUNEL staining of implanted tumor sections. Scale bar, 50μm. (B) Activation and cleavage of caspase-9, -3 in implanted tumor tissue. The figure shows the representative results of three independent repeated experiments. The data show the mean ± standard error of three independent repeated experiments (n=5-12). ***P<0.001, NS no significant difference (P>0.05).

[0050] Fig. 9ICAM-1 protein blocking antibody JH12 can effectively inhibit the growth of non-small cell lung cancer implants in mice. (A) BLI assay was used to detect the binding of JH12 antibody to ICAM-1 protein. (B) Functional assay of JH12 antibody inhibiting the binding of FG protein to A549 cells. Data show the mean ± standard deviation of three independent repeated experiments (n = 3). (C) Immunoblotting assay was used to detect the expression of apoptosis signal-related protein caspase-9 / 3 in A549 cells after treatment with JH12 antibody or human IgG protein. (D) Flow cytometry was used to detect the apoptosis of A549 cells after treatment with JH12 antibody or human IgG protein. Data show the mean ± standard deviation of three independent repeated experiments (n = 3). (E) Left panel, experimental design. Right panel, tumor growth in tumor-bearing mice after treatment with JH12 antibody or human IgG protein. Data show the mean ± standard error of three independent repeated experiments (n = 10-20). (F) TUNEL staining of implanted tumor sections. Scale bar, 50 μm. The figure shows the representative results of three independent repeated experiments. ***P<0.001, NS, no significant difference (P>0.05).

[0051] Fig.10 The relationship between the expression of ICAM-1 and FGG and the overall survival of patients with colorectal cancer, pancreatic cancer, and renal cancer. (A) Data from http: / / kmplot.com / analysis / . (B) Expression of ICAM-1 on the surface of normal lung bronchial epithelial cells 16HBE and different non-small cell lung cancer, colorectal cancer, pancreatic cancer, and renal cancer cell lines. The left figure shows the representative results of three independent repeated experiments. The data show the mean ± standard deviation of three independent repeated experiments (n = 3).

[0052] Fig.11 Detection of apoptosis in colon adenocarcinoma cells, pancreatic cancer cells, and renal cancer cells after knockdown of ICAM-1. (A, C, and E) ICAM-1 knockdown efficiency on the surface of colon adenocarcinoma cells SW480, HT-29, HCT 116, pancreatic cancer cells PANC-1, and renal cancer cells 769-P and 786-O. The left figure shows the representative results of three independent repeated experiments. The data show the mean ± standard deviation of three independent repeated experiments (n=3). (B, D, and F) Detection of apoptosis after knockdown of ICAM-1 in colon adenocarcinoma cells SW480, HT-29, HCT 116, pancreatic cancer cells PANC-1, and renal cancer cells 769-P and 786-O. The left figure shows the representative results of three independent repeated experiments. The data show the mean ± standard deviation of three independent repeated experiments (n=3). ***P<0.001. DETAILED DESCRIPTION

[0053] Intercellular cell adhesion molecule 1 (ICAM-1) belongs to the immunoglobulin superfamily and is a single-pass transmembrane glycoprotein. The expression of ICAM-1 is generally upregulated in tumor tissues. Clinical data analysis found that the expression level of ICAM-1 is negatively correlated with the prognosis of patients with non-small cell lung cancer, but the function of ICAM-1 in the occurrence and development of lung cancer is still unclear. The present invention discovered the role and regulatory mechanism of ICAM-1 on the survival and apoptosis of non-small cell lung cancer cells. Compared with normal lung epithelial cells, ICAM-1 is highly expressed in a variety of non-small cell lung cancer cell lines, and knocking down the expression of ICAM-1 in lung cancer cells can cause lung cancer cell apoptosis. Further studies have found that ICAM-1 on the surface of lung cancer cells maintains the normal survival signal of lung cancer cells by interacting with the fibrinogen γ chain (FGG) secreted by cancer cells. Mechanistically, the use of ICAM-1Δ8-22 amino acid deletion mutation (ICAM-1 8-22 is the FGG binding sequence) to disrupt the ICAM-1–FGG interaction will lead to a decrease in the phosphorylation level of the intracellular segment of ICAM-1, thereby disrupting the binding of the phosphatase SHP-2 to the tail of ICAM-1, further causing the phosphorylation level of ERK1 / 2 and Akt to be downregulated and the phosphorylation level of JNK and p38 MAPK to be increased, generating a series of apoptotic signals and caspase-9 / 3-dependent apoptotic pathways, ultimately leading to the apoptosis of non-small cell lung cancer cells A549 and H1650. Intervention of the ICAM-1 pathway will inhibit tumor progression in lung cancer-bearing mice. In addition, the ICAM-1–FGG signal may have similar survival regulatory effects on a variety of solid tumors other than non-small cell lung cancer, including colorectal cancer, pancreatic cancer, and renal cancer. Inhibiting the expression of ICAM-1 will significantly induce apoptosis of a variety of colorectal cancer, pancreatic cancer, and renal cancer cell lines.

[0054] Explanation of terms

[0055] In the present invention, the amino acid sequence of FGγ chain (FGG) (117-133) is: NNQKIVNLKEKVAQLEA, SEQ ID NO: 16; the amino acid sequence of ICAM-1 (8-22) is: KVILPRGGSVLVTCS, SEQ ID NO: 17.

[0056] In the present invention, the letters in the amino acid sequence represent the single-letter abbreviations of amino acids known in the art, such as those described in J.Biol.Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0057] In the present invention, the amino acid sequences of the above-listed CDRs are all shown in accordance with the definition of IMGT rules. However, it is well known to those skilled in the art that the CDR of an antibody can be defined in the art by a variety of methods, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be appreciated by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions as defined by any of the above-mentioned known schemes described in the present invention.

[0058] Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment system rules or combinations). Although the scope of the present invention is based on the sequences shown in the IMGT rule definition, the amino acid sequences corresponding to the definition rules of other CDRs should also fall within the scope of protection of the present invention.

[0059] In the present invention, the term "full-length antibody" is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in the present invention) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in the present invention) and a light chain constant region (abbreviated as CL in the present invention). The light chain constant region consists of one domain CL. Mammalian heavy chains are classified as α, δ, ε, γ and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins comprising α, δ, ε, γ and μ heavy chains are classified as immunoglobulins (Ig) A, IgD, IgE, IgG and IgM. Complete antibodies form a "Y" shape. The stem of the Y consists of the second and third constant regions of the two heavy chains (and the fourth constant region for IgE and IgM) bound together, and disulfide bonds (interchain) are formed in the hinge. The heavy chains γ, α and δ have a constant region consisting of three tandem (in a row) Ig domains, and a hinge region for increased flexibility; the heavy chains μ and ε have a constant region consisting of four immunoglobulin domains. The second and third constant regions are called "CH2 domains" and "CH3 domains", respectively. Each arm of the Y includes the variable region of a single heavy chain bound to the variable and constant regions of a single light chain and the first constant region. The variable regions of the light and heavy chains are responsible for antigen binding.

[0060] In the present invention, the "Fab" fragment consists of a light chain and the CH1 and variable region of a heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. The "Fc" region contains two heavy chain fragments including the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain. The "Fab'" fragment contains a light chain and a portion of a heavy chain including the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule. The "F(ab')2" fragment contains two light chains and two heavy chains including a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody, but lacking a constant region.

[0061] In the present invention, the scFv (single chain antibody fragment) may be a conventional single chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15 to 20 amino acids. The VL and VH domains are paired to form a monovalent molecule by enabling them to be produced as a linker for a single polypeptide chain [see, for example, Bird et al., Science 242: 423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)]. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers are composed of repeated G4S amino acid sequences or variants thereof. For example, a linker having an amino acid sequence (G4S)4 or (G4S)3 may be used, but variants thereof may also be used.

[0062] As known in the art, "nucleic acid" in the present invention refers to a nucleotide chain of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0063] For the purposes of this article, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that permits expression of mRNA, protein, polypeptide or peptide by a host cell when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide, and the vector contacts the cell under conditions sufficient to allow mRNA, protein, polypeptide or peptide to be expressed in the cell. The vector of the present invention is generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vector of the present invention may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthesized or partially obtained from a natural source, and which may contain natural, non-natural or altered nucleotides. The recombinant expression vector may comprise naturally occurring or non-naturally occurring nucleotides, or both types of connections. In exemplary aspects, altered nucleotides or non-naturally occurring nucleotides are connected without hindering transcription or replication of the vector.

[0064] The recombinant expression vector of the present invention can be any suitable recombinant expression vector, which can be used for transformation or transfection to deliver one or more genes or sequences of interest into any suitable host cell and preferably express the gene or sequence in the host cell. Suitable vectors include those designed for expansion and amplification or for expression or both, examples of vectors include but are not limited to viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0065] As used herein, the term "host cell" refers to any type of cell that can contain a nucleic acid or vector described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, an animal, a fungus, or an alga; or it can be a prokaryotic cell, such as a bacterium or a protozoa. In exemplary aspects, as described herein, the host cell is a cell originating from or obtained from an individual. In exemplary aspects, the host cell is derived from or obtained from a mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to rodents (order Rodentia) mammals, such as mice and hamsters; and lagomorpha (order Lagomorpha) mammals, such as rabbits. Preferably, the mammal belongs to the order Primate, the New World Monkeys (Ceboids) or the Lemurs (Simoids) or to the order Anthropoids. Particularly preferred mammals are humans.

[0066] The antibody can be obtained from the culture (eg, purified and isolated), and the methods for culturing the transformant and obtaining the antibody from the culture are both routine in the art.

[0067] In some embodiments, the pharmaceutical composition or pharmaceutical preparation of the present invention comprises suitable pharmaceutically acceptable carriers such as pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. As used in the present invention, "pharmaceutically acceptable carriers" or "pharmaceutical carriers" include any and all solvents, dispersion media, isotonic agents and absorption delay agents that are physiologically compatible. Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. For the use of excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, RC Rowe, PJ Eskey and SCOwen, Pharmaceutical Press, London, Chicago. If desired, the composition may also contain a small amount of wetting agent or emulsifier, or pH buffer. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Oral formulations may include standard pharmaceutical carriers and / or excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin. The pharmaceutical preparation or pharmaceutical composition comprising the present invention may be prepared by mixing the antibody or antigen-binding fragment thereof of the present invention with the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution. The pharmaceutical composition or preparation of the present invention can also include more than one active ingredient, which is required for the specific indication to be treated, preferably those active ingredients with complementary activities that do not adversely affect each other. For example, it is desirable to also provide other active ingredients, such as other antibodies, anti-tumor active agents, small molecule drugs or immunomodulators, etc. The active ingredients are suitably combined in an amount effective for the intended use. Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing antibodies of the present invention, and the matrix is ​​in the form of shaped articles, such as films or microcapsules.

[0068] In the present invention, an antibody-drug conjugate (ADC) is a small molecule drug with biological activity connected to a monoclonal antibody through a chemical chain, and the monoclonal antibody acts as a carrier to transport the small molecule drug into target cells. For example, the cytotoxic agent is MMAF or MMAE, or the label is a fluorescent agent.

[0069] Chimeric antigen receptor (CAR) is an engineered transmembrane protein that combines the specificity of antigen-specific antibodies with T cell receptor functions. In general, CAR includes an extracellular domain, a transmembrane domain, and an intracellular domain. In exemplary aspects, the extracellular domain of CAR includes an antigen recognition region, which can be an scFV of an antigen-specific antibody.

[0070] In the present invention, the application scenarios of "non-diagnostic purpose" and "non-therapeutic purpose" include but are not limited to: detecting the presence or absence of a protein containing the ICAM-1 8-22 antigen polypeptide in the laboratory; or using the antibody as described in one of the technical solutions of the present invention as a positive antibody to screen other antibodies that can inhibit the binding of intercellular adhesion molecule-1 to fibrinogen; or competing with other antibodies that can inhibit the binding of intercellular adhesion molecule-1 to fibrinogen to detect whether there is competition between antibodies, that is, whether the antigenic epitopes are the same or similar; or after culturing cancer cells that rely on ICAM-1-FGG interaction to maintain survival in vitro in the laboratory, treating them with a blocker that inhibits ICAM-1-FGG interaction to induce apoptosis, etc.

[0071] The "ICAM-1-FGG interaction" mentioned in the present invention refers to the fact that after ICAM-1 binds to FGG, it regulates the phosphorylation of the intracellular segment of ICAM-1 and recruits the phosphatase SHP-2 to bind to the tail domain of ICAM-1, thereby maintaining the activation of cell survival-related kinases such as ERK and Akt, while inhibiting the phosphorylation of JNK and p38MAPK, ultimately maintaining the survival and proliferation signals of cancer cells.

[0072] The "blocker for inhibiting ICAM-1-FGG interaction" of the present invention can specifically target the site of ICAM-1-FGG interaction and block the occurrence of the interaction, thereby causing the intracellular segment of ICAM-1 to undergo dephosphorylation and lose the binding of SHP-2, inhibiting the activation of key cell survival kinases ERK and Akt, significantly upregulating the activation of stress and apoptosis-related kinases such as JNK and p38 MAPK, and cancer cells initiate apoptosis programs dependent on the caspase-9 / 3 signaling pathway, ultimately affecting the survival of tumor cells in vitro and progression in vivo. The blocker of the present invention can be a small molecule chemical active substance or a biological macromolecule (such as a protein).

[0073] Since the present invention discovered the interaction between ICAM-1 and its binding protein fibrinogen γ chain that activates intracellular apoptosis signals in cancer cells, and revealed the molecular basis of the interaction and its regulatory mechanism for cancer cell apoptosis signals, a person skilled in the art can determine based on the description of the present invention that as long as the cancer cells rely on the ICAM-1–FGG interaction to maintain their survival, they can be induced to undergo apoptosis after the ICAM-1–FGG interaction is blocked.

[0074] Since the present invention discloses the regulatory mechanism of blocking ICAM-1–FGG interaction on cancer cell apoptosis signals, those skilled in the art can reasonably expect, based on the description of the present invention, that antibodies or polypeptides that specifically target and inhibit ICAM-1–FGG interaction can achieve the effect of inducing cancer cell apoptosis.

[0075] Since the present invention reveals that blocking the ICAM-1–FGG interaction can serve as a potential molecular target for treating a variety of cancers, a person skilled in the art can determine based on the description of the present invention that, as long as the cancer cells of the cancer rely on the ICAM-1–FGG interaction to maintain survival, they can be diagnosed, prevented and / or treated by blocking the ICAM-1–FGG interaction.

[0076] Since the present invention discloses that blocking ICAM-1–FGG interaction can be used as a potential molecular target for treating various cancers, those skilled in the art can reasonably expect, based on the present invention, that antibodies or polypeptides that specifically target and inhibit ICAM-1–FGG interaction can achieve the effect of being used in the preparation of drugs for diagnosing, preventing and / or treating cancer.

[0077] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0078] Experimental Materials and Methods

[0079] The antibodies used in the present invention are shown in Table 1, the reagents used are shown in Table 2, and the cell lines used are shown in Table 3.

[0080] 1.1 Materials

[0081] 1.1.1 Antibodies

[0082] Table 1

[0083]

[0084]

[0085] 1.1.2 Reagents

[0086] Table 2

[0087] name Purchased from Cell Counting Kit-8(C0038) Beyotime Annexin V-FITC / PI Apoptosis Detection Kit(A211-02) Vazyme One Step TUNEL Apoptosis Assay Kit(C1086) Beyotime

[0088] 1.1.3 Cell lines

[0089] Table 3

[0090] name Culture medium 16HBE DMEM / 10%FBS / L-Glu A549 DMEM / 10%FBS / L-Glu NCI-H358 1640 / 10%FBS / L-Glu NCI-H522 1640 / 10%FBS / L-Glu NCI-H1650 1640 / 10%FBS / L-Glu NCI-H1299 1640 / 10%FBS / L-Glu HCC827 1640 / 10%FBS / L-Glu SW480 1640 / 10%FBS / L-Glu HT-29 McCoy's 5A / 10%FBS / L-Glu HCT 116 McCoy's 5A / 10%FBS / L-Glu PANC-1 DMEM / 10%FBS / L-Glu 769-P 1640 / 10%FBS / L-Glu 786-O 1640 / 10%FBS / L-Glu

[0091] 1.2 Methods

[0092] 1.2.1 Calcium phosphate method for transient transfection of 293T cells

[0093] 1) Split cells one day before transfection so that the cells reach a density of 60-70% at the time of transfection.

[0094] 2) 1 hr before transfection, the culture medium was changed to Dulbecco's Modified Eagle Medium (DMEM) (Gibco, catalog number: 12100-046) containing 25 μM chloroquine, with a volume of 10 ml per 10 cm dish.

[0095] 3) In a 15 ml sterile centrifuge tube, add 10 μg of DNA, dissolve 1095 μl of sterilized MilliQ, and then add 155 μl of 2M CaCl2.

[0096] 4) Add 1,250 μl 2×HBS dropwise, mixing gently.

[0097] 5) Add the mixture dropwise directly to the cells, spreading evenly over the entire surface of the dish.

[0098] 6) Incubate at 37°C for 7-11 hr, rinse once, and replace with chloroquine-free culture medium.

[0099] 7) Collect cells 48-72 hr after transfection.

[0100] 1.2.2 Virus packaging and cell infection

[0101] Virus packaging:

[0102] 1) Split 293T cells the day before and the density should reach about 70% on the next day when transfected.

[0103] 2) Using the calcium phosphate transfection method, the plasmids for packaging the virus were transferred into 293T cells at a certain ratio.

[0104] 3) After 48 to 72 hours, collect the culture medium (containing the virus) and filter it with a 0.45 μm syringe filter before use.

[0105] 4) If necessary, ultracentrifuge to concentrate the virus at 27,000 rpm and 4°C for 2 hr.

[0106] Virus infected cells:

[0107] 5) Subculture the cells to be infected the day before.

[0108] 6) Mix the cell culture medium, virus and polybrene (final concentration 8 μg / ml) together and add to the cells.

[0109] 7) After 18 hours, remove the virus-containing culture medium and add fresh culture medium to continue culturing.

[0110] 1.2.3 Knockdown of ICAM-1 and fibrinogenγchain expression in lung cancer cells

[0111] According to the instructions of pLKO.1 vector (Addgene, catalog number: 24150), the relevant vectors were constructed and lentivirus was used to infect A549 or H1650 cells to knock down the expression of ICAM-1 and fibrinogenγchain. The short hairpin RNA (shRNA) related sequences used are as follows:

[0112] ICAM-1 (shICAM-1–1(SEQ ID NO:1):5'-GGAGCAAGACTCAAGACATGA-3'; shICAM-1–2(SEQ ID NO:2):5'-CCGGTATGAGATTGTCATCAT-3');

[0113] fibrinogenγchain (shFGG-1 (SEQ ID NO: 3): 5'-CCGGTGGTATTCCATGAAGAA-3'; shFGG-2 (SEQ ID NO: 4): 5'-CTGCATCTTAGATGAAAGAT-3');

[0114] control scrambled shRNA (SEQ ID NO: 5) (5′-CCTAAGGTTAAGTCGCCCTCG-3′).

[0115] The knockdown efficiency was detected 48 hours after virus infection.

[0116] 1.2.4 CCK-8 cell proliferation and toxicity assay

[0117] 1) Before cell treatment, add 200 μl of 5,000 cells to each well of a 96-well plate, and add 20 μl of CCK-8 solution to each well at different time points of shRNA treatment. Use the wells with the corresponding amount of cell culture medium and CCK-8 solution but no cells as blank controls.

[0118] 2) Continue incubating in the cell culture incubator for 2 hours.

[0119] 3) Measure the absorbance at 450 nm.

[0120] 1.2.5 Cell apoptosis detection

[0121] Sample staining:

[0122] 1) Induce cell apoptosis according to the experimental requirements. The test samples should include untreated cell samples as negative controls. In addition, when performing flow cytometry, the experimental groups should be stained with Annexin V-FITC and propidium iodide (PI) for compensation.

[0123] 2) Collect cells: Collect 1-5×10 5 Cells

[0124] Suspended cells: centrifuge at 1000 rpm, 4°C for 5 min, and discard the culture supernatant;

[0125] Adherent cells: Digest the cells with trypsin without EDTA, collect the cells after terminating the digestion, centrifuge at 1000 rpm and 4°C for 5 min, and discard the supernatant.

[0126] 3) Washing cells: Wash the cells twice with pre-cooled PBS, centrifuge at 1000 rpm, 4°C for 5 min each time, and discard the supernatant.

[0127] 4) Resuspend cells: Add 100 μl 1× Binding Buffer and gently blow to make a single cell suspension.

[0128] 5) Cell staining: Add 5μl Annexin V-FITC and 5μl PI Staining Solution, gently blow evenly; incubate at room temperature (20-25°C) for 10 min in dark; add 400μl 1×Binding Buffer, gently mix evenly. After staining, the sample should be detected by flow cytometry within 1 hour.

[0129] Sample Analysis:

[0130] The excitation wavelength of the flow cytometer is 488nm; the green fluorescence of FITC is detected in the FL1 channel; the red fluorescence of PI is detected in the FL2 or FL3 channel, and FL3 is recommended. 10,000 events are collected for each sample. Data analysis is performed using FlowJo and other software, with FL1 as the horizontal axis and FL3 as the vertical axis. The positive and negative boundaries of the two fluorescence parameters are determined based on the FITC and PI fluorescence values, and a cross gate is drawn. In a typical experiment, cells can be divided into three subpopulations: live cells are double negative (Annexin V-FITC - / PI - ); Early apoptotic cells are single positive for Annexin V-FITC (Annexin V-FITC + / PI - ); Late apoptotic cells are double positive for Annexin V-FITC and PI (Annexin V-FITC + / PI + ).

[0131] 1.2.6 Immunoprecipitation and Western Blot Analysis

[0132] Wash the cell surface with pre-cooled TBS, scrape the cells with a cell scraper, and dissolve them in pre-cooled TBS. Centrifuge at 3,000rpm for 3min, discard the supernatant, resuspend with Lysis buffer, and place on ice for 30min; centrifuge at 12,000rpm for 15min to obtain cell lysate. Protein content was determined according to the BCA method. Add specific antibodies to the cell lysate and incubate overnight at 4℃, use anti-rabbit IgG as a control, and then add protein G beads and incubate at 4℃ for 2hr. After washing the beads three times, prepare the sample.

[0133] Equal amounts of protein samples were separated by 9% SDS-PAGE electrophoresis; after electrophoresis, the protein was transferred to a nitrocellulose membrane (NC) using a wet electrotransfer instrument, with the current set to 280 mA and the time set to 80 min; after transfer, the NC membrane was removed, with the membrane containing the transferred protein facing up, and incubated with blocking solution (TBST containing 5% skim milk powder) at room temperature for 2 hours; incubated with specific antibodies at 4°C overnight; washed the NC membrane three times with TBST, each for 5 minutes; incubated with HRP-coupled secondary antibodies at room temperature for 1 hour; washed the NC membrane twice with TBST, each for 5 minutes. Then, fluorescent development was performed with ECL detection reagent.

[0134] 1.2.7 Establishment of mouse tumor model

[0135] 1) The state of cells is the key to tumorigenesis experiments, so the cell growth state must be good. Take cells in the logarithmic growth phase, and the cell density should be around 80-90%. Replace the culture medium with fresh one night before collecting cells.

[0136] 2) After trypsin digestion, wash the cells twice with pre-cooled PBS to remove serum from the cells.

[0137] 3) Use PBS or serum-free medium to blow the cell pellet to an appropriate concentration. The number of cells inoculated in subcutaneous tumors is 2×10 6 cells / tube, the inoculation volume is 0.1 ml, so the concentration of the cell suspension is 2×10 7 cells / ml.

[0138] 4) After the cells are digested, they should be inoculated subcutaneously into nude mice as soon as possible, generally within half an hour. During the process, the cell suspension should be placed on ice to reduce cell metabolism.

[0139] 5) The selected nude mice are generally 5-8 weeks old and weigh about 18-20g. The implantation site is selected in an area with rich blood supply, such as the middle and posterior part of the armpit and the middle and upper part of the groin.

[0140] 6) Use a gun to fully disperse the cell suspension before inoculation to prevent the cells from agglomerating and reducing the cell survival rate. During inoculation, the needle should be inserted a little deeper, about 1 cm deep, under the skin to reduce the cell suspension from overflowing from the needle hole after injection. Use the thumb and index finger of the left hand to pinch the skin on the back of the nude mouse's neck, and the little finger to clamp the nude mouse's tail.

[0141] 7) About a week after inoculation, a lump can be seen forming under the skin. Depending on the characteristics of the tumor cells and the amount of cells inoculated, the period of subcutaneous tumor formation is generally 1-2 months. The tumor should not grow too large, generally not exceeding 1000mm 3 Tumor size is usually measured twice a week, with a vernier caliper measuring the longest and shortest parts of the tumor. Volume = 0.5 × length × width 2 .

[0142] 1.2.8 TdT-mediated dUTP nick-end label (TUNEL) staining to detect cell apoptosis

[0143] 1) Fix tissue sections with 4% paraformaldehyde for 30-60 minutes.

[0144] 2) Wash twice with PBS or HBSS, 10 minutes each time.

[0145] 3) Add PBS containing 0.5% Triton X-100 and incubate at room temperature for 5 minutes.

[0146] 4) Prepare TUNEL detection solution: Each sample contains 5μl TdT enzyme and 45μl fluorescent labeling solution, and mix thoroughly. Note: The prepared TUNEL detection solution must be used once and cannot be frozen.

[0147] 5) Wash twice with PBS or HBSS.

[0148] 6) Add 50 μl of TUNEL detection solution to the sample and incubate at 37°C in the dark for 60 minutes.

[0149] 7) Wash 3 times with PBS or HBSS.

[0150] 8) After sealing with anti-fluorescence quenching sealing liquid, observe under a fluorescence microscope. The excitation wavelength range that can be used is 450-500nm, and the emission wavelength range is 515-565nm (green fluorescence).

[0151] 1.2.9 Phage display experiment

[0152] The ICAM-1 8-22 antigen polypeptide 100 μg / ml was coated on an immunotube, and the Tomlinson I+J phage library was subjected to three rounds of affinity enrichment screening of adsorption-elution-amplification in the immunotube. During the three rounds of enrichment screening, the intensity of nonspecific washing was gradually increased to fully remove nonspecifically bound phages. After each round of elution and amplification of bound phages, the next round of screening was carried out. In the fourth round, the purified ICAM-1 protein 20 μg / ml was used as an antigen, and the fourth round of adsorption and elution was carried out. Finally, the phages obtained from the fourth round of elution were infected with TG1 (OD=0.4-0.6) Escherichia coli strains. Single clones were randomly picked from the TYE plate coated in the fourth round to 96-well cell culture plates, and the phage supernatants of the single clones were prepared for phage ELISA detection.

[0153] 1.2.10 Bio-layer Interferometry (BLI)

[0154] BLI experiments were performed using an Octet Red 96 instrument (ForteBio, Inc.). The principle is to fix the biotinylated ICAM-1 protein on a streptavidin (SA) biosensor and incubate it with antibodies at different concentration gradients in a kinetic buffer to observe the dynamic binding and dissociation of the two. According to the overall fitting of the ICAM-1 and antibody binding curves at different concentration gradients to a 1:1 Langmuir binding model, R 2The KD value of the binding affinity of JH12 antibody to ICAM-1 was calculated when the value was ≥0.95. The binding experiment was performed at 25°C. The data were analyzed using Octet Data Analysis Software 9.0 (ForteBio, Menlo park, CA, USA).

[0155] 1.2.11 Functional detection of JH12 antibody inhibiting the binding of ICAM-1 to FG

[0156] Different dilutions of JH12 antibody were used to incubate A549 cells in 50ul PBS system at 4℃ for 30min; then 100nM Alexa Fluor 488-labeled FG protein was added and incubated at room temperature for 30min. The cells were washed twice and analyzed by flow cytometry. The same concentration of humanized IgG and Alexa Fluor 488-labeled FG protein incubation group was used as a negative control.

[0157] Example 1 ICAM-1 is highly expressed in non-small cell lung cancer cells and high expression of ICAM-1 is negatively correlated with the prognosis of lung cancer patients

[0158] Lung cancer is one of the most lethal cancer types in the world, with a 5-year survival rate of approximately 15%. Unlike normal lung epithelial cells, overactivation and upregulation of cancer cell survival signals often occur in lung cancer cells. Therefore, in clinical practice, lung cancer treatment is often accompanied by enhanced drug resistance of cancer cells, which seriously affects the survival prognosis of lung cancer patients. Therefore, it is extremely important to study the survival mechanism of lung cancer cells. According to its clinical pathological classification, it mainly includes two subtypes: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The former accounts for about 85% of all lung cancer cases and is currently the main type of lung cancer incidence and research. NSCLC can be further divided into lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). By comparing and analyzing 17 common malignant tumor types in The Cancer Genome Atlas (TCGA) database, it was found that the expression of ICAM-1 was significantly enriched in NSCLC samples ( Figure 1 A). At the same time, further analysis of the protein expression database Human Protein Atlas (HPA) revealed that the immunohistochemical staining results of 24 NSCLC pathological sections showed that most non-small cell lung cancer samples showed moderate to strong ICAM-1 staining signals ( Figure 1In addition, gene set enrichment analysis (GSEA) of sequencing data of tumor tissues of NSCLC cases in the TCGA database (including 519 LUAD and 497 LUSC cases) found that the expression level of ICAM-1 was positively correlated with the expression of key molecules in signaling channels related to the pathogenicity and development of non-small cell lung cancer ( Figure 1 C), which suggests that ICAM-1 molecules play a very important role in the occurrence and development of non-small cell lung cancer.

[0159] In order to further clarify the relationship between ICAM-1 and the occurrence and development of lung cancer, we first analyzed the existing tumor clinical database (data from http: / / kmplot.com / analysis / ). The results showed that the progression-free survival rate and overall survival rate of patients with non-small cell lung cancer with high expression of ICAM-1 were significantly lower than those with low expression ( Figure 1 D). The survival analysis results indicate that high expression of ICAM-1 in lung cancer patients plays a very important regulatory role in the occurrence and development of cancer. Next, we will further explore the function of ICAM-1 in the tumorigenesis process. First, the expression of ICAM-1 on the surface of normal lung bronchial epithelial cells 16HBE and non-small cell lung cancer cell lines NCI-H358, NCI-H522, A549, NCI-H1650, NCI-H1299 and HCC827 was compared. The results of flow cytometry showed that compared with normal lung epithelial cells 16HBE, the expression of ICAM-1 in lung cancer cells A549, NCI-H1650, NCI-H1299 and HCC827 was upregulated to varying degrees ( Figure 2 ). It was found that ICAM-1 was not only overexpressed to varying degrees in a variety of non-small cell lung cancer cell lines, but also negatively correlated with the prognosis of non-small cell lung cancer patients.

[0160] Example 2 Knockdown of ICAM-1 expression in non-small cell lung cancer cells induces cell apoptosis

[0161] In order to further study the function of ICAM-1 in lung cancer, among the various lung cancer cell lines overexpressing ICAM-1 mentioned above, the most widely studied A549 cells and the H1650 cells with higher ICAM-1 expression were selected as the research subjects for subsequent experiments. The control shRNA (control shRNA) or shRNA targeting ICAM-1 was transfected into the normal lung epithelial 16HBE and non-small cell lung cancer cell lines A549 and H1650 using the lentiviral system to achieve specific knockdown of ICAM-1 expression in 16HBE, A549 and H1650 cells ( Figure 3 The effect of knocking down ICAM-1 expression on lung cancer cell survival was detected ( Figure 3 B). The results of flow cytometry showed that the expression of ICAM-1 on the surface of cells transfected with ICAM-1shRNA-1 and shRNA-2 was significantly downregulated ( Figure 3 A). Annexin V-FITC / PI staining was then used to detect the proportion of cell apoptosis. In cells with early apoptosis, membrane phosphatidylserine PS flips from the inside of the cell membrane to the surface of the cell membrane and is exposed to the extracellular environment. Annexin V can bind to the cell membrane of early apoptotic cells through the phosphatidylserine exposed on the outside of the cell. Propidium iodide (PI) is a nucleic acid dye that cannot penetrate the intact cell membrane, but due to the increased cell membrane permeability of cells in the middle and late stages of apoptosis and dead cells, PI can penetrate the cell membrane and stain the cell nucleus red. Therefore, the combination of Annexin V and PI can distinguish cells at different stages of apoptosis. After normal lung epithelial cells 16HBE, non-small cell lung cancer cells A549 and H1650 were transfected with control shRNA and two ICAM-1 shRNAs, the cells treated with control shRNA and ICAM-1 shRNA were collected on days 4-6 for Annexin V-FITC / PI staining. The results showed that obvious apoptosis occurred in lung cancer cells A549 and H1650 in which ICAM-1 expression was knocked down. Among them, the proportion of A549 cells in the early stage of apoptosis was about 13.6%, and the proportion of cells in the late stage of apoptosis was about 7.76%. The proportion of H1650 cells in the early stage of apoptosis was about 17.4%, and the proportion of cells in the late stage of apoptosis was about 5.49%. However, since the expression of ICAM-1 in normal lung epithelial cells 16HBE was not high, there was no apoptosis after knocking down ICAM-1. And when the expression of ICAM-1 was restored in lung cancer cells A549 and H1650 in which ICAM-1 was knocked down, the apoptosis of lung cancer cells was significantly inhibited ( Figure 3 B). The above results indicate that ICAM-1 expressed by non-small cell lung cancer cells plays a significant regulatory role in cell survival.

[0162] Example 3 Fibrinogen γ chain (FGG) is an ICAM-1 ligand protein involved in regulating apoptosis signals in non-small cell lung cancer cells

[0163] Knocking down ICAM-1 expression in A549 cells and H1650 cells can cause apoptosis of lung cancer cells. It was found that ICAM-1 is very important for maintaining the survival of non-small cell lung cancer cells, but has no effect on the survival of normal lung epithelial cells. As an important adhesion molecule on the surface of epithelial cells, ICAM-1 can bind to a variety of ligands and transmit signals. Currently known ligands include integrin αLβ2, integrin αMβ2, mucin MUC1, and fibrinogen. In order to further explore the protein molecules involved in regulating the ICAM-1 survival signal in lung cancer cells, the expression of the above-mentioned reported ICAM-1 ligands in non-small cell lung cancer cells was first detected.

[0164] Flow cytometry was used to detect the expression of β2 integrin on the surface of 16HBE cells, A549 cells and H1650 cells. The results showed that β2 integrin was not expressed on the surface of 16HBE cells, A549 cells and H1650 cells. β2 integrin, as an important surface adhesion molecule mediating lymphocyte migration, was highly expressed on the surface of T cell line Jurkat cells ( Figure 4 A). MUC1, as a ligand of ICAM-1, is also overexpressed in a variety of tumor cells, and different cells have different splice forms of MUC1. Studies have found that only the full-length form of MUC1 / TR (tandem repeat domain) contains the VNTR repeat region in its N-terminal SEA domain. VNTR is the binding site of the first immunoglobulin domain of ICAM-1, while other splice forms lack the VNTR region and therefore cannot bind to ICAM-1. Immunoblotting antibodies that recognize different sites of MUC1 were selected to detect the expression of MUC1 in A549 cells and H1650 cells. The results showed that both A549 cells and H1650 cells only expressed short splice forms that could not bind to ICAM-1, but did not express the full-length form of MUC1 ( Figure 4 In B), human breast cancer cell line MCF7 was used as a positive control expressing the full-length MUC1 protein.

[0165] Next, the expression of fibrinogenγ chain in 16HBE cells, A549 cells and H1650 cells was detected by immunoblotting. The binding of fibrinogenγ chain to ICAM-1 was verified by co-IP experiment. The results showed that both A549 cells and H1650 cells expressed fibrinogenγ chain, and there was interaction between ICAM-1 and fibrinogenγ chain in A549 cells and H1650 cells ( Figure 4C). To further explore the effect of the combination of fibrinogen and ICAM-1 on the survival of lung cancer cells, shRNA targeting the fibrinogenγ chain was transfected into A549 and H1650 cells to knock down the expression of the fibrinogenγ chain, and then the knockdown efficiency of shFGG was verified by immunoblotting. The results showed that after A549 and H1650 cells were infected with shFGG lentivirus, the expression of the fibrinogenγ chain in the cells was significantly decreased ( Figure 4 D). Then, the apoptosis ratio of lung cancer cells with knockdown of fibrinogenγ chain was also detected by Annexin V-FITC / PI staining experiment. It was found that the loss of FGG function in non-small cell lung cancer cells would cause obvious cell apoptosis, which was similar to the result of ICAM-1 loss of function ( Figure 4 D and Figure 3 B).

[0166] When the ICAM-1Δ8-22 mutant protein lacking the FGG binding region was supplemented in the ICAM-1 knockdown non-small cell lung cancer cells A549 and H1650, it could not reverse the apoptosis caused by the loss of ICAM-1 function in lung cancer cells like the wild-type ICAM-1 protein was supplemented ( Figure 5 ). Restoring the expression of ICAM-1 intracellular segment deletion mutant protein (ICAM-1ΔCyto) will hinder the normal intracellular signal transduction of ICAM-1 and induce cell apoptosis. The above experiments confirm that the direct interaction between ICAM-1 and its ligand fibrinogenγ chain in non-small cell lung cancer cells is an important regulator of cell survival signals.

[0167] Example 4 Knockdown of ICAM-1 expression in non-small cell lung cancer cells leads to activation of apoptosis signals

[0168] Through the knockdown of ICAM-1 and ICAM-1 complementation experiments, and the effect on cell survival was detected, it was found that knockdown of ICAM-1 expression induced apoptosis of A549 cells and H1650 cells. Next, we further explored how ICAM-1 regulates intracellular signal-mediated cell survival, and explored the pathway of apoptotic signals mediated by ICAM-1 through the study of classical apoptotic signals. The generation of different apoptotic signals will activate caspase-8 or caspase-9. As the initiator of cell apoptosis, caspase-8 or caspase-9 can further activate downstream executor caspase proteins such as caspase-3, caspase-6 or caspase-7, thereby mediating cell apoptosis. The experimental results show that knockdown of ICAM-1 expression can cause the activation of caspase-9 and caspase-3, and has no effect on the caspase-8 apoptotic signal. At the same time, after the ICAM-1 knockdown lung cancer cells A549 and H1650 were supplemented with the ICAM-1Δ8-22 mutant protein lacking the FGG binding region, it could not reverse the apoptosis caused by the loss of ICAM-1 function in lung cancer cells like the wild-type ICAM-1 protein, while the wild-type ICAM-1 protein could reverse this phenomenon ( Figure 6 A).

[0169] The interaction between ICAM-1 and FGG on the surface of epithelial cells will cause tyrosine phosphorylation of the intracellular segment of ICAM-1. The phosphorylated intracellular segment of ICAM-1 can then recruit the phosphatase SHP-2, thereby affecting subsequent intracellular kinase pathways, transcriptional regulation, and cell survival signals. It was found that in the non-small cell lung cancer cells A549 and H1650 in which ICAM-1 was knocked down, the complementation of the ICAM-1Δ8-22 mutant protein lacking the FGG binding segment significantly inhibited the tyrosine phosphorylation level of the intracellular segment of ICAM-1, and the binding of the phosphatase SHP-2 was also significantly reduced; while the complementation of the wild-type ICAM-1 did not affect the phosphorylation of the intracellular segment of ICAM-1 and the recruitment of SHP-2. At the same time, when the ICAM-1 protein with a missing intracellular segment (ICAM-1ΔCyto) was complemented, the binding of the phosphatase SHP-2 was significantly inhibited due to the obstruction of intracellular signal transduction, and the transmission of downstream survival signals was further affected ( Figure 6 B). The above experiments indicate that the ICAM-1–FGG interaction in NSCLC cells will directly affect the intracellular signal transduction of ICAM-1.

[0170] The results showed that after knocking down ICAM-1 in non-small cell lung cancer cells A549 and H1650, the phosphorylation levels of Akt (Ser473) and ERK1 / 2 (Thr202 / Tyr204) were significantly downregulated, while the phosphorylation levels of JNK (Thr183 / Tyr185) and p38 (Thr180 / Tyr182) were significantly upregulated ( Figure 6 C). After complementation of the ICAM-1Δ8-22 mutant protein lacking the FGG binding region in the ICAM-1 knockdown non-small cell lung cancer cells A549 and H1650, it was not able to reverse the changes in the intracellular signaling pathway caused by the loss of ICAM-1 function in lung cancer cells like complementation of the wild-type ICAM-1 protein, while complementation of the wild-type ICAM-1 could reverse this phenomenon ( Figure 6 C). The above experimental results indicate that the functional loss of ICAM-1 in lung cancer cells will directly affect the phosphorylation of its intracellular segment and the subsequent kinase signaling, thereby affecting the transcriptional regulation and survival of the cells, and this regulation is directly dependent on the interaction between ICAM-1 and FGG.

[0171] Example 5 Inhibition of ICAM-1-FGG interaction affects the growth of non-small cell lung cancer tumors in mice

[0172] In order to further study the effect of ICAM-1–FGG signaling on the occurrence and development of non-small cell lung cancer in vivo, the non-small cell lung cancer cell line A549 transfected with control shRNA, ICAM-1 and FGG specific shRNA, and ICAM-1 knockdown and restored the expression of ICAM-1Δ8-22 mutant protein with FGG binding site deletion and wild-type ICAM-1 protein were implanted subcutaneously on the ventral side of immunodeficient nude mice to observe the growth of non-small cell lung cancer implanted tumors in different treatment groups. Starting from the day of tumor implantation, the growth of subcutaneous tumors was monitored every 3 days, and tumor-bearing mice were euthanized on the 15th day and tumor samples were collected. Subcutaneous tumor sample detection found that the levels of ICAM-1 and FGG expression in the tumor decreased significantly after transfection with ICAM-1 and FGG specific shRNA ( Figure 7 ).

[0173] After transfection with ICAM-1 and FGG-specific shRNA, tumor growth slowed significantly. Subcutaneous tumors in mice lacking ICAM-1 or FGG showed obvious spontaneous tumor regression on the 6th day and shrank rapidly within 15 days after inoculation ( Figure 8A, B), indicating that ICAM-1–FGG signaling plays an important regulatory role in the growth and progression of non-small cell lung cancer in vivo. The volume of A549 subcutaneous tumors with knockdown of ICAM-1 or FGG expression was significantly reduced compared with the control group tumors or subcutaneous tumors that restored expression of wild-type ICAM-1 protein, and restoring expression of ICAM-1Δ8-22 mutant protein lacking FGG binding sites did not change the delayed growth and regression of subcutaneous tumors caused by the loss of ICAM-1 ( Figure 8 These experimental results are consistent with the in vitro apoptosis of non-small cell lung cancer cells A549 and H1650 induced by ICAM-1 or FGG deficiency, confirming that ICAM-1–FGG interaction is directly involved in regulating the survival and progression of non-small cell lung cancer.

[0174] When cells undergo apoptosis, some DNA endonucleases are activated, which cut the genomic DNA between nucleosomes. When cells undergo apoptosis, DNA is extracted and electrophoresed, and a 180-200bp DNA ladder can be found. When the genomic DNA breaks, the exposed 3'-OH can be added with dUTP (fluorescein-dUTP) labeled with the green fluorescent probe fluorescein (FITC) under the catalysis of terminal deoxynucleotidyl transferase (TdT), so that it can be detected by fluorescence microscopy or flow cytometry. This is the principle of TUNEL (TdT-mediateddUTP Nick-End Labeling) method for detecting cell apoptosis. By performing TUNEL staining on each group of tumor samples, it was found that tumor samples lacking ICAM-1 or FGG expression showed obvious TUNEL + signal, and complementation of the ICAM-1Δ8-22 mutant protein lacking the FGG binding site could not restore the apoptosis caused by the loss of ICAM-1 or FGG as well as complementation of the wild-type ICAM-1 protein ( Figure 8 C). In addition, the apoptotic signaling in non-small cell lung cancer caused by the loss of ICAM-1–FGG interaction is also caspase-9 / 3 signaling dependent ( Figure 8 D), these experimental results are consistent with the conclusions of the in vitro apoptosis experiment of non-small cell lung cancer cell lines.

[0175] Example 6 Screening for antibodies that inhibit the binding of ICAM-1 to FGG

[0176] The above study revealed that non-small cell lung cancer cells can maintain the survival signal of tumor cells by binding to ICAM-1 on the cell surface through secreted FGG. Therefore, based on this mechanism, antibodies that inhibit the binding of ICAM-1 protein to FGG were screened, thereby specifically inhibiting the survival and growth of non-small cell lung cancer. In order to screen antibodies that block the binding of ICAM-1 and FGG, phage display technology was used to screen scFv and construct full-length antibodies. First, the Tomlinson I+J phage library was subjected to four rounds of affinity enrichment screening. The first three rounds used ICAM-1 8-22 polypeptide as antigen, and the last round used full-length ICAM-1 protein as antigen. After four rounds of screening, two scFvs that bind to ICAM-1 protein and ICAM-1 8-22 polypeptide were finally screened, named JH12 and JE1, respectively, and their amino acid sequences are shown below. The variable region sequences of the light and heavy chains were further constructed into the human IgG1 antibody framework for expression and purification.

[0177] JH12 antibody light chain CDR1 (SEQ ID NO: 6): QSISSY;

[0178] JH12 antibody light chain CDR2: RAS;

[0179] JH12 antibody light chain CDR3 (SEQ ID NO: 7): QQTDHMPLT;

[0180] JH12 antibody heavy chain CDR1 (SEQ ID NO: 8): GFTFSSYA;

[0181] JH12 antibody heavy chain CDR2 (SEQ ID NO: 9): IPPSGVRT;

[0182] JH12 antibody heavy chain CDR3 (SEQ ID NO: 10): AKGLRKFDY;

[0183] Among them, the above CDR region of the JH12 antibody is defined by the IMGT rules.

[0184] JH12 antibody light chain variable region (SEQ ID NO: 11):

[0185] DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASRLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTDHMPLTFGQGTKVEIKR;

[0186] JH12 antibody heavy chain variable region (SEQ ID NO: 12):

[0187] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIPPSG VRTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGLRKFDYWGQGTLV TVSS;

[0188] JH12 humanized chimeric antibody light chain sequence (SEQ ID NO:13):

[0189] MRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYRASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTDHMPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0190] JH12 humanized chimeric antibody heavy chain sequence (SEQ ID NO:14):

[0191] MDWTWRILFLVAAATGAHSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIPPSGVRTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGLRKFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0192] JE1 antibody scFv sequence (SEQ ID NO: 15):

[0193] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIGKNGIKTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRRPPFDYWGQGTLVTVSSGGGG SGGGGSGGGGSTDIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASLLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRSFRPMTFGQGTKVEIKR.

[0194] Since the full-length JE1 antibody failed to recognize and effectively bind to the non-small cell lung cancer cell line expressing ICAM-1 protein, the functional identification of the JH12 monoclonal clone was performed next.

[0195] Example 7 Functional identification of JH12 antibody

[0196] In order to further study the physiological function of JH12 antibody, the affinity between JH12 antibody and ICAM-1 protein was detected by small molecule kinetic experiment based on the principle of biomembrane interference. Finally, the dissociation constant of JH12 antibody was calculated to be 9.15nM ( Fig. 9 A). Next, A549 cells were treated with different concentrations of JH12 antibody, and the mixture was incubated with FG protein labeled with AlexaFluor 488. The half-inhibitory concentration of JH12 antibody to inhibit the binding of FG protein to A549 cells was detected by flow cytometry. The results showed that the half-inhibitory concentration of JH12 antibody was 0.89μg / ml ( Fig. 9 In addition, it was found that JH12 antibody can induce activation of apoptosis-related caspase-9 / 3 protein ( Fig. 9 C) and apoptosis of A549 cells ( Fig. 9 In addition, the JH12 antibody was found to effectively inhibit the growth of A549 subcutaneous tumors using a mouse lung cancer subcutaneous tumor model ( Fig. 9 E) and induce apoptosis of tumor cells ( Fig. 9 F).

[0197] Example 8 Knockdown of ICAM-1 expression induces apoptosis in colorectal cancer, pancreatic cancer and renal cancer cells

[0198] Considering the important role of ICAM-1–FG signaling in regulating apoptosis of non-small cell lung cancer cells, we explored the response of other solid tumors with high expression of ICAM-1 to the inhibition of ICAM-1 signaling. By analyzing the tumor clinical database (data from http: / / kmplot.com / analysis / ), it was found that in addition to non-small cell lung cancer, the expression of ICAM-1 and FGG was also negatively correlated with the overall survival rate of patients with colorectal cancer, pancreatic cancer and renal cancer ( Fig.10 A).

[0199] Next, a series of colorectal cancer, pancreatic cancer and renal cancer cell lines were selected to explore the effect of ICAM-1 expression on cell survival, including: colon adenocarcinoma cells SW480, HT-29 and HCT 116; pancreatic ductal adenocarcinoma cells PANC-1; renal cancer cells 769-P and 786-O ( Fig.10 B). The results showed that when ICAM-1 shRNA was used to treat the above cancer cells, different colorectal cancer cells, pancreatic cancer cells and renal cancer cells all underwent significant apoptosis compared with the control scrambled shRNA treatment group ( Fig.11 ). The experimental conclusion is consistent with the conclusion that the loss of ICAM-1 will significantly cause apoptosis of non-small cell lung cancer cells. The above experimental results suggest that ICAM-1-FG signaling not only has a significant role in maintaining growth and inhibiting apoptosis for non-small cell lung cancer, but may also have similar regulatory effects on other solid tumors expressing ICAM-1. Specific inhibition of ICAM-1-FG signaling may have certain therapeutic significance for various tumor types including non-small cell lung cancer.

Claims

1. An anti-ICAM-1 antibody, characterized in that The antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3; wherein: The amino acid sequence of the LCDR1 is shown in SEQ ID NO:6, the amino acid sequence of the LCDR2 is RAS, the amino acid sequence of the LCDR3 is shown in SEQ ID NO:7, the amino acid sequence of the HCDR1 is shown in SEQ ID NO:8, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:9, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:

10.

2. The antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region is shown in SEQ ID NO:11; and / or, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

12.

3. The antibody according to claim 1 or 2, characterized in that The antibody is a full-length antibody, Fab, Fab', F(ab')2 or Fv; Preferably, the Fv is a scFv.

4. The antibody according to claim 3, characterized in that The antibody is a full-length antibody, and its heavy chain constant region and / or light chain constant region are derived from a human antibody; Preferably, the heavy chain constant region and / or light chain constant region is derived from a human IgG1 antibody; More preferably, the amino acid sequence of the light chain of the antibody is shown as SEQ ID NO:13; and / or, the amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO:

14.

5. An isolated nucleic acid, characterized in that The nucleic acid encodes the antibody according to any one of claims 1 to 4.

6. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid of claim 5.

7. A transformant, characterized in that: The transformant comprises the nucleic acid according to claim 5 or the recombinant expression vector according to claim 6, and the host cell of the transformant is a eukaryotic cell or a prokaryotic cell.

8. A method for preparing an anti-ICAM-1 antibody, characterized in that: The method comprises culturing the transformant according to claim 7.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

10. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the antibody according to any one of claims 1 to 4.

11. A genetically modified cell, characterized in that The genetically modified cell comprises the chimeric antigen receptor of claim 10; Preferably, the genetically modified cells are eukaryotic cells, preferably isolated human cells; more preferably immune cells such as T cells or NK cells.

12. An antibody-drug conjugate, characterized in that: The antibody-drug conjugate comprises a cytotoxic agent or a label, and the antibody according to any one of claims 1 to 4.

13. A kit, characterized in that: The kit comprises the antibody according to any one of claims 1 to 4, the pharmaceutical composition according to claim 9, the chimeric antigen receptor according to claim 10, the genetically modified cell according to claim 11 and / or the antibody-drug conjugate according to claim 12.

14. A medicine kit, characterized in that: The kit comprises kit A and kit B, wherein: The drug kit A contains the antibody according to any one of claims 1 to 4, the pharmaceutical composition according to claim 9, the chimeric antigen receptor according to claim 10, the genetically modified cell according to claim 11 and / or the antibody-drug conjugate according to claim 12; The medicine kit B contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and / or other anti-tumor drugs.

15. A drug delivery device, characterized in that: The drug delivery device comprises the antibody according to any one of claims 1 to 4, the pharmaceutical composition according to claim 9, the chimeric antigen receptor according to claim 10, the genetically modified cell according to claim 11 and / or the antibody-drug conjugate according to claim 12; Preferably, the drug delivery device further comprises a component for administering the antibody, the chimeric antigen receptor, the antibody-drug conjugate, the genetically modified cell or the pharmaceutical composition to a subject, such as a syringe or an infusion device.

16. A method for immunodetection or determination of a protein containing an ICAM-1 8-22 antigen polypeptide, characterized in that: The method comprises using the antibody according to any one of claims 1 to 4, the pharmaceutical composition according to claim 9, the chimeric antigen receptor according to claim 10, the genetically modified cell according to claim 11 and / or the antibody-drug conjugate according to claim 12; the method is for non-diagnostic purposes; Preferably, the protein comprising the ICAM-1 8-22 antigen polypeptide is ICAM-1.

17. Use of the antibody according to any one of claims 1 to 4, the pharmaceutical composition according to claim 9, the chimeric antigen receptor according to claim 10, the genetically modified cell according to claim 11 and / or the antibody drug conjugate according to claim 12 in the preparation of a preparation for inhibiting the binding of a protein containing an ICAM-1 8-22 antigen polypeptide and a protein containing a fibrinogen γ chain; Preferably, the protein comprising the ICAM-1 8-22 antigen polypeptide is ICAM-1 and / or the protein comprising the fibrinogen γ chain is fibrinogen.

18. A method for causing apoptosis of cancer cells, characterized in that: The method comprises treating cancer cells with a blocker that inhibits ICAM-1–FGG interaction and inducing apoptosis in cancer cells, wherein the cancer cells rely on ICAM-1–FGG interaction for survival; the method is for non-therapeutic purposes; Preferably, the cancer cells are lung cancer cells, colorectal cancer cells, pancreatic cancer cells or renal cancer cells; the lung cancer cells are preferably non-small cell lung cancer cells, and the colorectal cancer cells are preferably colon adenocarcinoma cells; and / or, the inhibitor for inhibiting ICAM-1–FGG interaction is an antibody, a polypeptide or a small molecule drug, a chimeric antigen receptor comprising the antibody, an antibody-drug conjugate, a genetically modified cell or a pharmaceutical composition, or a pharmaceutical composition comprising the polypeptide or small molecule drug; More preferably, the non-small cell lung cancer cells are A549, H1650, H1299 or HCC827, the colon adenocarcinoma cells are SW480, HT-29 or HCT 116, the pancreatic cancer cells are PANC-1, and the renal cancer cells are 769-P or 786-O; and / or, the blocker for inhibiting ICAM-1–FGG interaction is the antibody according to any one of claims 1 to 4, the pharmaceutical composition according to claim 9, the chimeric antigen receptor according to claim 10, the genetically modified cells according to claim 11, or the antibody-drug conjugate according to claim 12.

19. Use of a blocker that inhibits ICAM-1–FGG interaction in the preparation of a drug for diagnosing, preventing and / or treating cancer, wherein the cancer cells rely on ICAM-1–FGG interaction to survive; Preferably, the cancer is lung cancer, colorectal cancer, pancreatic cancer or renal cancer; the lung cancer is preferably non-small cell lung cancer, and the colorectal cancer is preferably colon adenocarcinoma; and / or the inhibitor for inhibiting ICAM-1–FGG interaction is an antibody, a polypeptide or a small molecule drug, a chimeric antigen receptor comprising the antibody, an antibody-drug conjugate, a genetically modified cell or a pharmaceutical composition, or a pharmaceutical composition comprising the polypeptide or small molecule drug; More preferably, the inhibitor of ICAM-1–FGG interaction is the antibody according to any one of claims 1 to 4, the pharmaceutical composition according to claim 9, the chimeric antigen receptor according to claim 10, the genetically modified cell according to claim 11 or the antibody-drug conjugate according to claim 12.

Images