WO2022171195A1

WO2022171195A1 - Treatment of gastric cancer by using anti-cd87 antibody in combination with anti-pd1 antibody

Info

Publication number: WO2022171195A1
Application number: PCT/CN2022/076086
Authority: WO
Inventors: 焦作义; 王�琦; 秦龙; 孙辉; 王龙; 王彦; 张俊昶; 杨志良; 文飞; 蔡伟文
Original assignee: 兰州大学第二医院; 兰州华帜天成生物科技有限责任公司
Priority date: 2021-02-11
Filing date: 2022-02-11
Publication date: 2022-08-18
Also published as: WO2022171196A1; CN114920841A; CN114917329B; CN114917329A; CN114920841B

Abstract

Provided are a novel anti-CD87 antibody and a specific chimeric antigen receptor thereof, a CAR-T cell comprising the specific chimeric antigen receptor thereof, and use thereof in combination with an anti-PD 1 antibody in treatment of gastric cancer.

Description

Combination of anti-CD87 antibody and anti-PD1 antibody in the treatment of gastric cancer

technical field

The present invention relates to the fields of biology and medicine, in particular to a novel anti-CD87 antibody and its specific chimeric antigen receptor, CAR-T cells comprising its specific chimeric antigen receptor and its combined therapy with an anti-PD1 antibody Application in gastric cancer.

Background technique

Gastric cancer is one of the most common malignant tumors in human beings. There are more than 600,000 new cases of gastric cancer in my country every year, ranking second in the incidence of malignant tumors, seriously endangering human life and health. Due to the high heterogeneity of gastric cancer tissue, traditional treatment methods such as surgery, chemotherapy and radiotherapy are not satisfactory, and the five-year survival rate of advanced gastric cancer is less than 30%. It showed that patients with diffuse gastric cancer according to Lauren's classification had the worst clinical prognosis, and the 5-year overall survival rate was about 8% lower than that of intestinal gastric cancer patients. Therefore, the traditional treatment methods for gastric cancer are not effective, and it is urgent to develop new treatment models to improve their efficacy.

CAR-T therapy is a new way to treat tumors. Mainly used in hematological tumors. In 2017, the US FDA has approved two CAR-T cell drugs targeting CD19 for the treatment of leukemia and lymphoma. The progress of CAR-T therapy in the field of solid tumors is slow. The main reason is that CAR-T cells will be inhibited by the inhibitory tumor microenvironment (Tumor Microenvironment TME) after infiltrating into solid tumor tissue. Scientists express chemotaxis in CAR-T cells. factor receptors to enhance the migration ability and anti-tumor activity of CAR-T cells to tumors, or directly target proteins related to the tumor microenvironment. Therefore, research on CAR-T therapy targeting solid tumors and their microenvironment is a difficulty to be broken through in this field in the future.

There are many CAR-T therapies for gastric cancer. Chen Rin's group reported a study on the effect of CAR-T cells targeting HER2 in killing gastric cancer cells. The results showed that they had strong killing effects in vivo and in vitro, but the positive rate of HER2 in gastric cancer was low, and it was reported that it was only 5%. %about. The Li Peng group reported the study of gastric cancer CAR-T targeting mesothelin, which showed a good killing effect in the constructed NSI mice, and has not yet focused on gastric cancer. Li Zonghai's group developed a high-affinity humanized monoclonal antibody specific for Claudin18.2, and developed the second generation of CAR-T cells to study the antitumor activity in vitro and in vivo. T cells showed good antitumor activity in the human-derived gastric cancer PDX mouse model, and gastric cancer has not been specifically involved. In conclusion, CAR-T cell therapy for gastric cancer has good application prospects, however, no effective specific CAR-T cell therapy has been developed for gastric cancer with poor traditional therapy.

SUMMARY OF THE INVENTION

The inventors collected 15 pairs of cancer tissue and adjacent tissue samples that were pathologically diagnosed as gastric cancer, and screened the gastric cancer-specific protein CD87 by label-free quantitative proteomics. CD87, PLAUR or uPAR (which are used interchangeably herein), is a urokinase-type plasminogen activator (uPA) receptor that is anchored to cell membranes by GPIs and is involved in inflammation, tissue remodeling, and It is highly expressed in various cancers and has a poor prognosis. CD87 regulates the degradation of extracellular matrix proteins and activates multiple intracellular signaling pathways by cooperating with uPA, integrin and vitronectin, and plays an important role in tumor cell migration and proliferation. target. Our study found that CD87 is related to the tumor microenvironment. It not only participates in the degradation of extracellular matrix, but also promotes the infiltration of immune cells. It can be used as a target for CAR-T therapy in gastric cancer.

At present, preclinical studies and clinical trials of CAR-T for the treatment of gastric cancer emerge in an endless stream, but different CAR-T treatment strategies for different targets are still not ideal compared to the effect of CAR-T in the treatment of hematological tumors. However, CD87 is highly expressed on the surface of gastric cancer cells and is lowly expressed in normal tissues. Targeting CD87 can promote the degradation of extracellular matrix, improve the physical barrier of the tumor microenvironment, and promote immune cell infiltration. CAR-T is used as a target. The therapy can overcome the physical barrier of the tumor microenvironment and achieve better tumor killing effect.

Accordingly, in one aspect, the invention provides an anti-CD87 antibody comprising at least 90% (eg at least 91%, 92%, 93%, 94%, 95%, 96%) of SEQ ID NO: 10 or 14 , 97%, 98%, 99% or 100%) sequence identity to VH sequences and have at least 90% (e.g. at least 91%, 92%, 93%, 94%, 95%, VL sequences of 96%, 97%, 98%, 99% or 100%) sequence identity.

In a preferred embodiment, the anti-CD87 antibody comprises the VH sequence of SEQ ID NO: 10 or 14 and the VL sequence of SEQ ID NO: 12 or 16.

In another aspect, the invention provides an anti-CD87 antibody comprising at least 90% (eg at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) heavy chain sequence identity and have at least 90% (e.g. at least 91%, 92%, 93%, 94%, 95%, 96%) with SEQ ID NO: 4 or 8 %, 97%, 98%, 99% or 100%) sequence identity of the light chain.

In preferred embodiments, the anti-CD87 antibody comprises the heavy chain of SEQ ID NO: 2 or 6 and the light chain of SEQ ID NO: 4 or 8.

In another aspect, the present invention finds that anti-CD87 antibodies can be used to alter the tumor microenvironment. Accordingly, in one embodiment, the present invention provides the use of an anti-CD87 antibody for altering the tumor microenvironment. In a preferred embodiment, the anti-CD87 antibody is an anti-CD87 antibody as defined above.

In another aspect, the present invention provides the use of an anti-CD87 antibody as defined above for the treatment of gastric cancer in a patient, eg a human.

In another aspect, the present invention provides a CD87-specific chimeric antigen receptor (CAR) polypeptide comprising a CD87 antigen binding domain, a transmembrane domain and an intracellular signaling domain. In specific embodiments, the CD87 antigen binding domain comprises a variable heavy chain (VH) and a variable light chain (VL), the VH comprising at least 90% (eg, at least 90% of SEQ ID NO: 10 or 14) and a variable light chain (VL) 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity, the VL comprises a sequence with at least SEQ ID NO: 12 or 16 Sequences of 90% (eg, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity. In a preferred embodiment, VH comprises the sequence of SEQ ID NO: 10 or 14 and VL comprises the sequence of SEQ ID NO: 12 or 16.

For chimeric antigen receptors, see M. Sadelain, I. Rivière, R. Brentjens, Targeting tumors with genetically enhanced T lymphocytes. Nat. Rev. Cancer 3, 35-45 (2003).

In specific embodiments, the CD87-specific chimeric antigen receptor (CAR) polypeptide further comprises a signal peptide, such as the signal peptide IL2-SP (eg, the sequence encoded by SEQ ID NO: 18 or at least 80% identical thereto). sequences of sequence identity). In specific embodiments, the CD87-specific chimeric antigen receptor (CAR) polypeptide further comprises a linker polypeptide between the VH and VL of the CD87 antigen binding domain (eg, the sequence encoded by SEQ ID NO: 19 or sequences with at least 80% sequence identity). In specific embodiments, the CD87-specific chimeric antigen receptor (CAR) polypeptide further comprises a hinge region between the CD87 antigen-binding domain and the transmembrane domain, such as the hCD8a hinge (e.g., represented by SEQ ID NO: 20 encoded sequences or sequences having at least 80% sequence identity therewith). In specific embodiments, the transmembrane domain may be, for example, CD8tTM (eg, the sequence encoded by SEQ ID NO: 21 or a sequence having at least 80% sequence identity thereto). In specific embodiments, the intracellular signaling domain comprises a costimulatory domain and a signaling domain, the costimulatory domain may be, for example, a 4-1BB costimulatory domain (e.g., represented by SEQ ID NO: 22 encoded sequence or a sequence having at least 80% sequence identity therewith), the signaling domain may be, for example, a CD3zeta signaling domain (e.g., the sequence encoded by SEQ ID NO: 23 or a sequence having at least 80% sequence identity therewith) sexual sequence).

In another aspect, the present invention provides the use of a CD87-specific chimeric antigen receptor polypeptide as described above for altering the tumor microenvironment.

In another aspect, the present invention provides the use of a CD87-specific chimeric antigen receptor polypeptide as described above for the treatment of gastric cancer in a patient (eg, a human).

In another aspect, the present invention provides an isolated nucleic acid sequence encoding an antibody or chimeric antigen receptor polypeptide as described above.

In another aspect, the present invention provides a vector comprising the isolated nucleic acid sequence as described above.

In another aspect, the present invention provides a cell comprising the vector as described above. In specific embodiments, the cell is selected from the group consisting of: αβT cells, γδT cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, innate lymphocytes (ILCs), cytokine-induced killing (CIK) cells, cytotoxic T lymphocytes (CTL), lymphokine-activated killer (LAK) cells, regulatory T cells, or any combination thereof. In specific embodiments, the cell expresses a CD87-specific chimeric antigen receptor polypeptide as described above.

In another aspect, the present invention provides the use of a cell as described above for altering the tumor microenvironment.

In another aspect, the present invention provides the use of a cell as described above for the treatment of gastric cancer in a patient.

The inventors have also surprisingly found that by targeting CD87, the effect of PD-1 inhibitors in gastric cancer treatment can also be significantly improved. Without being bound by theory, it is believed that CD87 inhibitors alter the tumor microenvironment in the body by targeting CD87, thereby helping PD-1 inhibitors work better therapeutically. Such a combined effect, more particularly a synergistic effect, of a CD87 inhibitor and a PD-1 inhibitor would not have been expected by those skilled in the art.

As used herein, a CD87 inhibitor can be any agent that inhibits CD87. Preferably, the CD87 inhibitor is an anti-CD87 antibody as described above, a CD87-specific chimeric antigen receptor (CAR) polypeptide or a cell comprising a CD87-specific chimeric antigen receptor (CAR) polypeptide. As used herein, a PD-1 inhibitor can be any agent that inhibits PD-1. Preferably, the PD-1 inhibitor is an anti-PD-1 antibody.

Accordingly, in one aspect, the present invention provides an anti-CD87 antibody, a CD87-specific chimeric antigen receptor (CAR) polypeptide, or a cell comprising a CD87-specific chimeric antigen receptor (CAR) polypeptide for use in combination with a PD-1 inhibitor Use in the treatment of gastric cancer in a patient. In specific embodiments, the anti-CD87 antibody, CD87-specific chimeric antigen receptor (CAR) polypeptide or cell comprising a CD87-specific chimeric antigen receptor (CAR) polypeptide alters the tumor microenvironment in the patient. As used herein, a patient can be any mammalian or non-mammalian, preferably a human.

In preferred embodiments, the anti-CD87 antibody, CD87-specific chimeric antigen receptor (CAR) polypeptide or cell comprising a CD87-specific chimeric antigen receptor (CAR) polypeptide is an anti-CD87 antibody as described above , CD87-specific chimeric antigen receptor (CAR) polypeptide or cells comprising CD87-specific chimeric antigen receptor (CAR) polypeptide.

Description of drawings

Figure 1 shows the expression of CD87 provided by the present invention in gastric cancer and normal group. A: The expression level of PLAUR gene in gastric cancer patients was verified by qRT-PCR. The results showed that PLAUR was highly expressed in gastric cancer tissues and lowly expressed in adjacent tissues (P<0.05, n=12). B, C: Western Blot was used to verify the expression level of PLAUR protein in gastric cancer patients. The results showed that PLAUR was significantly expressed in gastric cancer tissues and low in adjacent tissues (P<0.01, n=8).

Figure 2 shows the expression level of CD87 in gastric cancer cell lines, A: qRT-PCR; B: flow cytometry. A: The expression level of PLAUR in gastric cancer cell lines (MKN-45, AGS, SNU-216, N87, HGC-27 and GES) was verified by qRT-PCR, and the results showed that PLAUR was highly expressed in AGS and SNU-216 cells. B: Flow cytometry was used to detect the expression level of PLAUR in gastric cancer cell lines (MKN-45, AGS, SNU-216, N87, HGC-27 and GES), and the results also showed that PLAUR was highly expressed in AGS and SNU-216 cells .

Figure 3 is a cell experiment: CD87 knockout inhibits tumor cell proliferation: A: SNU216; B: AGS. C, D: Knockout of PLAUR in SNU-216 cells and AGS cells, respectively, MTT results showed that knockdown of PLAUR inhibited tumor cell proliferation.

Figure 4 is an animal experiment. A: tumor volume change; B: tumor weight; C: small animal imaging. A: NSG mice were subcutaneously inoculated with MKN-45 cells, MKN-45-PLAUR ^-/- and MKN-45-PLAUR ^OE cells, respectively. The results showed that knockout of PLAUR in MKN-45 cells inhibited tumor cell proliferation (p< 0.05), reply PLAUR to restore the function. B: Mouse tumor tissue weight. C: Small animal imaging results.

Figure 5: Expression of extracellular matrix proteins after AGS and SNU-216 knockout PLAUR.

Figure 6 shows the preparation of CD87 monoclonal antibody (also known as anti-UPAR antibody): A: cellular immunity; B: positive B cell screening and antibody flow verification; C: two screened high-affinity monoclonal antibodies.

Figure 7 is a flow-through validation of CD87 monoclonal.

Figure 8: Anti-uPAR antibody competes with uPA for uPAR binding: (A) Binding affinity (KD) of anti-uPAR monoclonal antibody (mAb) to human uPAR was assessed by surface plasmon resonance. (B-C) Immunofluorescence (IF) (B) and immunoelectron microscopy (C) analysis of anti-uPAR binding to uPAR-expressing HEK-293T cells. (D-E) Detection of different forms of uPAR by anti-uPAR mAbs shown by flow cytometry (FCM) (D) and Western blotting (E). (F) Competitive binding assay showing that anti-uPAR mAb detected by FCM inhibits the binding of uPA to uPAR.

Figure 9: Anti-uPAR antibody inhibits uPAR-dependent signaling in gastric cancer cells (A) Phosphorylated ERK (p-ERK) and total ERK (p-ERK) in SNU-216 cells pretreated with anti-uPAR or ctrl mAb after pro-uPA stimulation T-ERK) Western blot. (B-E) Growth curves (B, C), transwell invasion assay (D) and cell adhesion assay (E) of AGS and SNU-216 cells treated with anti-uPAR or ctrl mAb. Data are presented as mean±SEM (*p<0.05, **p<0.01, ***p<0.001).

Figure 10 is a cell experiment: CD87 monoclonal antibody blocking inhibits tumor cell proliferation: A: AGS; B: SNU216

Figure 11 is the data of CD87 monoclonal antibody killing CDX model.

Figure 12 shows the changes of tumor tissue weight and mouse weight in CDX model group killed by CD87 monoclonal antibody.

Figure 13 shows the imaging results of CD87 monoclonal antibody killing the CDX model of small animals.

Figure 14: Anti-uPAR alone or in combination with anti-PD-1 improves survival in humanized mice bearing patient-derived xenografts. (A) Diagram of the experimental procedure. (B-C) Antibody-treated tumor growth (B) and survival curves (C) of mice bearing PDX from patients (n=5/group, 10 mg/kg/antibody, I.P., every 5 days starting at 8 dpi); expressed as Mean±SEM (ns not significant, *p<0.05, **p<0.01, ***p<0.001).

Figure 15: Anti-uPAR alone or in combination with anti-PD-1 enhances infiltration and activation of cytotoxic CD8+ T cells.

(AB) IF staining (A) and quantification (B) of CD8+ T cells in patient A-derived PDX at the end of the experiment. (CD) CD8 IHC staining and data analysis of the same PDX as in (AB). (E) mRNA expression levels of chemokines in PDX as in (AB) detected by qRT-PCR. (F) Activated cytotoxic T cells (CD45 ⁺ CD8 ⁺ CD107a ⁺ ), regulatory T cells (Tregs, CD45 ⁺ CD4 ⁺ FOXP3 ⁺ ) and Frequency of M2 macrophages (CD45 ⁺ CD11b ⁺ CD68 ⁺ CD206 ⁺ ) as indicated by FCM. Data are presented as mean±SEM (ns not significant, *p<0.05, **p<0.01, ***p<0.001).

Figure 16 is a schematic diagram of the construction of CD87-CAR.

Figure 17 is a schematic diagram of the structure of CD87-CAR of the present invention.

Fig. 18 is a graph showing the infection efficiency of T cells. The initial infection efficiency was 27.8%, and the infection efficiency reached 99.9% after sorting and enrichment.

Figure 19 shows the establishment of two groups of CD87-CAR-T+AGS-luciferase and control-T cells+AGS-luciferase for co-culture, and at 1:1, 2:1, 4:1, 8:1 and The luciferase release of tumor cells in each group was observed under the ratio of 16:1, and the line graph was drawn. (Effective cells: target cell binding/killing screening).

Figure 20 shows the CDX model established in NSG mice with MKN-45 cells, which are divided into T cell control group, CAR-T cell killing group and CAR-T cell combined with PD-1 antibody treatment group. A: Changes in tumor volume; B: Animal survival analysis.

Figure 21 shows the expression levels of IFN-γ and Granzyme B in the serum of each group of animals.

Figure 22 shows the imaging results of small animals.

Figure 23 shows the experimental results of CAR-T cells killing PDX model. They were divided into T cell control group, CAR-T cell killing group and CAR-T cell combined with PD-1 antibody treatment group. A: Changes in tumor tissue volume after treatment; B: Analysis of survival curve after treatment.

Figure 24 shows the expression levels of IFN-γ and Granzyme B in the serum of animals in each group.

Figure 25 shows the infiltration of CD8+ T cells in the combination group of CAR-T cells and PD-1 antibody.

Example

The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. For those without specifying the specific conditions in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

Example 1

CAR-T target screening and validation: A total of 605 up-regulated genes were identified in the cancer and adjacent tissues of 16 gastric cancer patients for gene expression profiling analysis. Fifteen pairs of fresh surgical samples of gastric cancer and adjacent tissues were collected and analyzed by label-free quantitative proteomics. A total of 186 proteins that were highly expressed in cancer and not expressed adjacent to cancer were identified. Using the TCGA gastric cancer data, the differential genes of diffuse gastric cancer and normal tissues were analyzed. The above two data obtained were intersected with immune-related genes, and two key proteins, FCGR3A and CD87, were finally obtained, which were analyzed in the TISIDB database with immune cell infiltration and immune cells. Checkpoint dependencies. According to the basic principle that the expression level of CAR-T membrane protein in normal human tissues and organs must be low, and the expression level in cancer tissues is high and related to its biological behavior, combined with the expression and distribution of CD87 in normal human tissues and organs, CD87 is preliminarily determined as a gastric cancer CAR -T therapeutic targets.

Example 2

The expression levels of CD87 in gastric cancer cell lines HGC-27, MKN-45, AGS, SNU-216, HS746T and NCI-N87 were detected by qRT-PCR and flow cytometry, and the results showed that CD87 in AGS, SNU-216 and HS746T High expression, low expression in HGC-27, MKN-45 and NCI-N87,

On this basis, the gRNA sequences of the gene CD87 to be knocked out were designed: gRNA1: TTCCACACGGCAATCCCCGT. gRNA2: GGACCACGATCGTGCGCTTG. Knockout of CD87 in AGS and SNU-216 showed that knockout of CD87 in AGS and SNU216 inhibited tumor cell proliferation, and antibody blocking achieved the same effect, using 2 × 10 ⁶ MKN-45 cells, MKN -45-CD87 ^-/- cells and CD87-reverting MKN-45-CD87 ^-/- cells formed tumors in NSG mice, and tumor volumes in mice were measured every two days, and the tumor-bearing mice were observed with a small animal imager. Tumor changes.

CD87 can change the tumor microenvironment by detecting the changes of ECM-related adhesion proteins. It is found that the expression of adhesion-related proteins such as Smad2, vitronectin and vimetin is significantly down-regulated, which makes the intercellular adhesion become loose, which may be related to the changes in the tumor microenvironment.

Example 3

Preparation of CD87 monoclonal antibody

Preparation of CD87 monoclonal antibody to construct lentiviral plasmid Lenti-CMV-CD87-T2A-GFP:

The constructed plasmids and packaging plasmids pDD and pVSVg were transfected with PEI at a ratio of 1:1:0.5 to 293T cells. After culturing for 36 hours, the supernatant was collected, centrifuged and concentrated the virus. After measuring the titer, 293T cells were infected with puromycin. The cells stably expressing the antigen CD87 were screened and successfully obtained.

Experimental steps:

1.1 Use Gibson ligation system to design primers to amplify the CD87 sequence and GFP sequence respectively, and the PCR products are recovered by gel.

1.2 The Lenti-CMV-Puro plasmid BamHI was digested, and the digested product was recovered by gel.

1.3 Use Gibson ligation system to connect, transform, extract plasmids for sequencing and expression verification.

1.4 Those identified correctly were named Lenti-CMV-CD87-T2A-GFP plasmid.

Construction of 293T cell line stably expressing CD87: 293T-CD87:

Experimental Materials

Lentiviral vector plasmid Lenti-CMV-CD87-T2A-GFP; packaging plasmid: pDD, pVsVG; 293T cells; 0.25% trypsin; DMEM; FBS; Opti-MEM; lip2000; BSA.

Experimental procedure

1) Inoculation of 293T cells: Digest 293T cells in good growth state with 0.25% trypsin, adjust the cell concentration to 6×10 ⁵ cells/mL, and inoculate 10 mL in a 10 cm petri dish, incubate at 37°C and 5% carbon dioxide for 18 hours , so that the cell confluency reaches 60%-70%, and the culture medium is changed to Opti-MEM half an hour before transfection.

2) Virus packaging: Add 7ug Lenti-CMV-CD87-T2A-GFP plasmid, 5ug pDD and 3.5ug pVsVg to 500uL OPTI-MEM and mix well; add 30uL lip2000 to 500uL OPTI-MEM and mix well, let stand at room temperature for 5 minutes, slowly add it to the plasmid mixture and mix well, let it stand at room temperature for 15 minutes, add it dropwise to the petri dish, and mix well. After 6 hours, it was replaced with fresh DMEM medium containing 10% FBS and 1% BSA.

3) Virus concentration: after culturing for 60 hours, collect the supernatant, centrifuge at 3000rpm at 4°C for 10 minutes, filter with a 0.45um filter membrane, and ultracentrifuge at 25000rpm at 4°C for 2 hours, discard the supernatant, and use 300uL containing 10% FBS and 1% BSA The pellet was resuspended in DMEM medium, overnight at 4°C, aliquoted in 50uL/tube, quick-frozen on dry ice and stored at -80°C.

4) Infect 293T cells with lentivirus to construct a 293T cell line stably expressing CD87, named 293T-CD87.

Monoclonal antibodies targeting CD87 were prepared using the prepared 293T-CD87 cells.

293T-CD87 cells were used for cellular immunization: 4 female Balb/c mice of SPF grade 6-8 weeks were taken, the orbital blood was collected one day before immunization, and the serum was separated as a negative control. Mice were injected subcutaneously with 1×10 ⁷ 293T cells stably expressing CD87. The second and third immunizations were performed on

days

10 and 20 with 5 × 106 293T cells stably expressing CD87, respectively. On the 25th day, the orbital blood of mice was collected, and the titer of CD87 antibody was detected by ELISA. The mouse with the highest antibody titer was selected and injected with 2×10 ⁶ cells for booster immunization. On the 30th day, the spleen cells of the immunized mice were prepared, the positive B cells that could produce CD87 antibody were sorted by flow cytometry (FCM), and the VH and VL sequences were obtained and verified by single-cell RT-PCR technology. High affinity monoclonal antibodies against CD87 (A7 and E2).

Example 4

The monoclonal antibodies targeting CD87 prepared in Example 3 were characterized, and the prepared monoclonal antibodies A7 and E2 were verified by flow cytometry, WB and laser confocal respectively.

Example 5

The therapeutic effect of the monoclonal antibody targeting CD87 prepared in Example 3 on the CDX model of gastric cancer cell line and the therapeutic effect of combined PD-1.

NSG mice were subcutaneously injected with about 2×10 ⁶ MKN-45 cells overexpressing CD87-T2A-luciferase to establish a CDX model in mice. They were divided into control group, CD87 monoclonal antibody treatment group, PD-1 monoclonal antibody treatment group and CD87 monoclonal antibody treatment group. In the anti-PD-1 antibody group, when the tumor volume reached about 90 mm ³ , 2×10 ⁷ human PBMCs were injected into the tail vein, respectively, and 10 mg/kg CD87 monoclonal antibody, PD-1 monoclonal antibody, and CD87 monoclonal antibody were administered in the administration group, respectively. +PD-1 antibody, the control group was given the same dose of human IgG antibody twice a week. The tumor size of the mice was measured every two days, and the small animal in vivo imaging system was used to analyze and detect the changes of the tumor every 4 days to verify the therapeutic effect of CD87 monoclonal antibody.

Example 6

The therapeutic effect of the monoclonal antibody targeting CD87 prepared in Example 3 on the PDX model derived from gastric cancer patients and the therapeutic effect of combined PD-1.

Tumor tissues from patients with pathologically confirmed CD87+ gastric cancer were taken, cut into pieces and then transplanted into NSG mice subcutaneously. When the tumors grew, they were taken out. Part of the tumor tissues were cryopreserved, the other part was analyzed for gene expression, and the remaining part was inoculated into NSG mice. The third-generation tumors were not different and could be used for tumor killing experiments. Twenty model mice were prepared and divided into control group, CD87 monoclonal antibody treatment group, PD-1 monoclonal antibody treatment group and CD87 monoclonal antibody combined with PD-1 antibody group. 2×10 ⁷ human PBMCs were injected, the administration group was given 10 mg/kg CD87 monoclonal antibody, PD-1 monoclonal antibody and CD87 monoclonal antibody + PD-1 antibody respectively, and the control group was administered the same dose of human IgG antibody, twice a week. The tumor size of the mice was measured every two days, and the small animal in vivo imaging system was used to analyze and detect the changes of the tumor every 4 days to verify the therapeutic effect of CD87 monoclonal antibody.

Example 7

Preparation of CD87-CAR

Preparation of CD87-CAR plasmid, the CD87-CAR structure consists of EF1a promoter nucleic acid sequence, IL2 signal peptide, CD87 scFv sequence, hCD8a hinge region nucleic acid sequence, CD8TM transmembrane region nucleic acid sequence, 41-BB costimulatory signal domain nucleic acid sequence , CD3ζ signaling domains formed in series, named EF1a-CD87 scFv-hCD8a hinge-CD8TM-41-BB-CD3ζ. The nucleic acid sequence of the CD87-CAR gene structure constructed in this example is shown in SEQ ID No. 24 in the sequence listing.

Experimental steps:

1. Using the Gibson ligation system to design primers to amplify the CD87 scFv sequence, and recover the PCR product by gel.

2. The lenti-EF1a-hCAR-Puro plasmid was digested with EcoRI and BamHI, respectively, and the digested products were recovered by gel.

3. Use Gibson ligation system to connect, transform, extract plasmids for sequencing and expression verification.

4. The correct one is CAR plasmid, named CD87-CAR plasmid.

Packaging and Concentrating Lentivirus Using the Constructed CD87-CAR Plasmid

Experimental Materials

Lentiviral vector plasmid CD87-CAR; packaging plasmid: pMD2.G, pSPAX2; 293T cells; 0.25% trypsin; DMEM; FBS; Opti-MEM; lip2000; BSA.

Experimental procedure

1) Inoculation of 293T cells: Digest 293T cells in good growth state with 0.25% trypsin, adjust the cell concentration to 6 × 10 ⁵ cells/mL, and inoculate 10 mL in a 10 cm petri dish, incubate at 37°C and 5% carbon dioxide for 18 hours , so that the cell confluency reaches 60%-70%, and the culture medium is changed to Opti-MEM half an hour before transfection.

2) Virus packaging: Add 7ug CD87-CAR plasmid, 5ug psPAX2 and 3.5ug pMD2.G to 500uL OPTI-MEM and mix well; add 30uL lip2000 to 500uL OPTI-MEM and mix well, let stand for 5 minutes at room temperature, and add slowly Mix well into the plasmid mixture, let stand for 15 minutes at room temperature, add dropwise to the petri dish, and mix well. After 6 hours, it was replaced with fresh DMEM medium containing 10% FBS and 1% BSA.

Infection of human peripheral blood T cells with the provided lentivirus

Experimental Materials

EasySep ^™ Human T Cell Isolation Kit (StemCell, Catalog #17951); Dynabeads Human T-Activator CD3/CD28 (Life Technologies, Catalog number: 11131D); PBS; FBS; RMPI-1640; human IL-2; Lymphoprep (Catalog # 07801); SepMate-50 (Catalog #15450).

Experimental procedure

1) Preparation of human PBMC cells: Pipette 15 mL of lymphocyte separation solution (Lymphoprep, Catalog #07801) into a centrifuge tube (SepMate-50, Catalog #15450). The upper end of the lymphocyte separation solution should submerge the filter screen. Take 10-15mL of fresh plasma, dilute with an equal volume of 2% FBS-PBS, gently pipette and mix, keep the centrifuge tube vertical, add the mixed sample along the tube wall (the centrifuge on the filter is mixed with the sample), room temperature Centrifuge at 1200g for 10 minutes, pour out the supernatant liquid into a new centrifuge tube, and centrifuge at 300g for 8 minutes at room temperature (if necessary, wash once with 2% FBS-PBS). The obtained PBMCs were used for T cell sorting.

2) T cell sorting: add 0.5-2 mL of T cell isolation buffer to PBMC, and use EasySep ^TM Human T Cell Isolation Kit (StemCell, Catalog #17951) to sort T cells: add 2 mL of prepared PBMC to the flow tube, add 50uL /mL isolation Cocktail, mix well, room temperature for 5min, vortex RapidSpheres for 30s to mix well, add 40uL/mL, make up to 2.5mL, pipette 3 times to mix well. As for the 5min on the magnetic rack, carefully aspirate the supernatant into a new 5mL flow tube, place it on the magnetic rack again for 5min, carefully aspirate the supernatant and place it in a centrifuge tube, centrifuge the T cells with complete medium 10% FBS+ RMPI-1640+IL2+1%P/S culture.

3) T cell activation: add T cells to a 24-well plate, 2×10 ⁶ cells/well, add 50uL of washed Dynabeads Human T-Activator CD3/CD28 (4×10 ⁷ beads/mL): magnetic beads and T cells The cell ratio was 1:1, and the cells were incubated at 37°C for 24 hours in a CO2 incubator.

4) T cell infection: The activated T cells were activated for 24-48 hours, added with concentrated virus and 4.4ug/mL Polybrene, and centrifuged at 32°C for 90min (1000×g) for infection. (MOI=5-10)

5) T cell culture: 8-10 hours after centrifugation, the medium was removed by centrifugation, and all infected wells were combined and cultured with complete medium 10% FBS+RMPI-1640+IL2+1%P/S. The medium was changed every 48 hours.

6) CD87-CAR-T cell sorting and determination: After 6 days of culture, remove the magnetic beads (place on a magnetic rack for 1-2 min, transfer the supernatant to a new culture flask, continue to culture for 24 hours, repeat if necessary This step), GFP detection of CAR expression.

7) Cryopreservation of CD87-CAR-T cells: Cryopreservation of CAR-T cells in 10% DMSO-FBS, 3×10 ⁶ cells/tube.

8) Sequencing verification of CD87-CAR gene structure expression: Take 1×10 ⁶ CAR-T cells, extract the total cell RNA with TRIzol and reverse-transcribe it into cDNA, and amplify the fragment containing the CD87-CAR gene structure by PCR. The upstream primers are: 5'-GTGTCGTGATCTAGAGCTAGCGGCCACCATGTACAGGATGCAACTCCTG-3'; the downstream primer is: 5'-GACTTCCTCTGCCCTCAGCGGCCGCCCGAGGCGGCAGGGCCTGCATGTG-3'. The amplification results were sent to sequencing for identification.

Example 8

The CD87-CAR-T cells prepared in Example 7 were used to detect the killing effect of the CD87-CAR-T cells on tumor cell lines.

CD87-CAR-T cells were co-cultured with luciferase-overexpressing gastric cancer cell lines AGS, AGS-CD87 ^-/- and MKN-45 in a certain proportion. After co-culture at 37°C for 4 hours, 50uL of supernatant and prepared luciferin were taken. The enzyme reagent (Promina#E2610) was added to the 96-well plate (Corning#3904), and the release of luciferase in the supernatant was detected by a microplate reader and the killing ability was calculated. To verify the binding ability and killing effect of CD87-CAR-T cells on gastric cancer target cells.

Example 9

The CD87-CAR-T cells prepared in Example 7 were used to detect the killing effect of the CD87-CAR-T cells on the gastric cancer cell line CDX model.

NSG mice were subcutaneously injected with about 2 × 10 MKN ^- 45 cells overexpressing CD87-T2A-luciferase to establish a CDX model in mice, and they were divided into T cell control group, CD87-CAR-T cell treatment group and CD87-CAR-T cell treatment group In the combination group of cells and PD-1 antibody, when the tumor volume reached about 90mm ³ , 1×10 ⁶ T cells, CD87-CAR-T cells, CD87-CAR-T cells and PD-1 antibody were injected into the tail vein respectively. . The small animal in vivo imaging system was used to analyze the changes of tumors, and ELISA was used to detect the changes of IFN-γ and Granzyme B cytokines in peripheral blood to verify the ability of CD87-CAR-T cells to bind gastric cancer target cells in vivo and their killing effect.

Example 10

The CD87-CAR-T cells prepared in Example 7 were used to detect the killing effect of the CD87-CAR-T cells on the gastric cancer patient-derived PDX model.

Tumor tissues from patients with pathologically confirmed CD87+ gastric cancer were taken, cut into pieces and then transplanted into NSG mice subcutaneously. When the tumors grew, they were taken out. Part of the tumor tissues were cryopreserved, the other part was analyzed for gene expression, and the remaining part was inoculated into NSG mice. The third-generation tumors were not different and could be used for tumor killing experiments. Twenty model mice were prepared and divided into T cell control group, CD87-CAR-T cell treatment group, and CD87 ^- CAR-T cell and PD-1 antibody combination group. 1×10 ⁶ T cells, CD87-CAR-T cells, CD87-CAR-T cells and PD-1 antibody were injected intravenously, respectively. The body weight and tumor size of the mice were monitored, and the levels of cytokines such as IFN-γ and Granzyme B in peripheral blood were detected by ELISA to verify the ability of CD87-CAR-T cells to kill the gastric cancer PDX model in vivo.

Claims

Use of an anti-CD87 antibody, a CD87-specific chimeric antigen receptor (CAR) polypeptide, or a cell comprising a CD87-specific chimeric antigen receptor (CAR) polypeptide in combination with a PD-1 inhibitor for the treatment of gastric cancer in a patient.
The use of claim 1, wherein the anti-CD87 antibody, CD87-specific chimeric antigen receptor (CAR) polypeptide, or cells comprising a CD87-specific chimeric antigen receptor (CAR) polypeptide alter the tumor microenvironment in the patient.
The use of claim 1 or 2, wherein the anti-CD87 antibody comprises at least 90% (eg at least 91%, 92%, 93%, 94%, 95%, 96%, 97%) of SEQ ID NO: 10 or 14 , 98%, 99% or 100%) sequence identity to VH sequences and have at least 90% (e.g. at least 91%, 92%, 93%, 94%, 95%, 96%, VL sequences of 97%, 98%, 99% or 100%) sequence identity,

Preferably, it comprises the VH sequence of SEQ ID NO: 10 or 14 and the VL sequence of SEQ ID NO: 12 or 16,

Preferably, it comprises at least 90% (eg at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) with SEQ ID NO: 2 or 6 Heavy chains of sequence identity to SEQ ID NO: 4 or 8 having at least 90% (e.g. at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) %) light chains of sequence identity,

Preferably, it comprises the heavy chain of SEQ ID NO: 2 or 6 and the light chain of SEQ ID NO: 4 or 8.
The purposes of claim 1 or 2, wherein said chimeric antigen receptor (CAR) polypeptide comprises CD87 antigen binding domain, transmembrane domain and intracellular signaling domain,

Preferably, wherein the CD87 antigen binding domain comprises a variable heavy chain (VH) and a variable light chain (VL), the VH comprising at least 90% (eg at least 91%, eg at least 91%, SEQ ID NO: 10 or 14) 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity, the VL comprising at least 90% ( for example sequences of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity,

Preferably, VH comprises the sequence of SEQ ID NO: 10 or 14 and VL comprises the sequence of SEQ ID NO: 12 or 16.
The purposes of claim 1 or 2, wherein the cell is selected from the group consisting of: αβT cells, γδT cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, innate lymphocytes (ILC), cytokine-induced killing ( CIK) cells, cytotoxic T lymphocytes (CTL), lymphokine-activated killer (LAK) cells, regulatory T cells, or any combination thereof.