CN107126563B

CN107126563B - Composition containing low-dose antibody for blocking VEGF signal path and application thereof

Info

Publication number: CN107126563B
Application number: CN201610107589.4A
Authority: CN
Inventors: 杨林; 黄玉辉; 游凤涛
Original assignee: Perongen Biotherapeutics Suzhou Co ltd
Current assignee: Perongen Biotherapeutics Suzhou Co ltd
Priority date: 2016-02-26
Filing date: 2016-02-26
Publication date: 2021-09-10
Anticipated expiration: 2036-02-26
Also published as: CN107126563A

Abstract

The invention provides a composition containing a low-dose VEGF signal channel blocker and application thereof. Specifically, the invention provides the application of a low-dose VEGF signal pathway blocker in preparing a tumor immunotherapy accelerant, and a composition containing the low-dose VEGF signal pathway blocker. Experiments of the invention show that the VEGF signal pathway blocker with low dose can induce the normalization of tumor blood vessels, remarkably improve the effect of tumor immunotherapy and promote the infiltration of T lymphocytes to tumor tissues.

Description

Composition containing low-dose antibody for blocking VEGF signal path and application thereof

Technical Field

The invention relates to the field of medicine, in particular to a composition containing a low-dose VEGF signal pathway blocker, such as an antibody for blocking VEGF signal pathways, and application thereof.

Background

Folkman first reported in 1971 that solid tumors exceeded 2-3mm in volume³In time, the growth of tumor bodies cannot be supported by pure oxygen diffusion, and the growth of tumor bodies can be supported by the formation of new blood vessels. Compared with normal blood vessels, the tumor neovascularization has the phenomena of vasodilatation, tortuosity and saccular structure formation, generates irregularly connected branches, and has uneven blood vessel density distribution; the loss of the caliber adjusting mechanism leads to abnormal shunt, and the vascular permeability and the vascular clearance are both obviously increased. Different from each otherThe frequent tumor neovascularization finally causes uneven blood vessel distribution, blood flow stagnation in partial areas, aggravates the formation of tumor hypoxia microenvironment, further influences the tumor gene phenotype, activates angiogenesis factors including Vascular Endothelial Growth Factor (VEGF), promotes the generation of tumor angiogenesis, causes the instability of tumor cell genes, activates some tumor cell survival factors, causes the tolerance of tumor cells to radiotherapy and chemotherapy, and further promotes tumor metastasis. More importantly, tumor vessels lack High Endothelial Venules (HEVs) and thus insufficient T cell infiltration, rendering immunotherapeutic strategies ineffective.

The technology for treating malignant tumors by Chimeric Antigen Receptor (CAR) gene modified T lymphocytes (hereinafter referred to as CAR-T cells) is selected as the first scientific invention in 2013, the complete remission rate of over 90 percent is achieved in the treatment of relapsed and refractory acute lymphatic leukemia, and the technology representing the international leading and recent development trend is popularized to various cancer treatment tests including solid tumors. However, the homing of intravenously-infused CAR-T cells to the tumor foci is severely hampered and CAR-T cell survival inside the tumor is impaired due to the presence of dysfunctional tumor vessels and immunosuppressive tumor microenvironment, so that the effect of this revolutionary technology on solid tumor therapy is still far from ideal and CAR-T cell application to solid tumor therapy is still internationally challenging. Thus, there is an urgent need in the art to develop new, effective methods of treating solid tumors using CAR-T cells.

Disclosure of Invention

The invention aims to provide a CAR-T cell medicine composition for treating solid tumors based on tumor vessel normalization and intervention strategies.

In a first aspect of the present invention, there is provided a pharmaceutical composition for the treatment of solid tumors, comprising a first active ingredient and a second active ingredient, wherein the first active ingredient is a VEGF signaling pathway blocker and the second active ingredient is a tumor immunotherapeutic agent,

and the pharmaceutical composition is in unit dosage form, wherein the content of the first active ingredient in each unit dosage form is 0.1 to 0.5 (preferably 0.15-0.4, or 0.2-0.25) of the conventional dose, wherein the conventional dose is 200-400mg/50kg body weight.

In another preferred embodiment, the conventional dose is the total conventional dose of the first active ingredient for each administration, such as 200-400mg/50kg body weight.

In another preferred embodiment, said one unit dosage form comprises the first active ingredient in an amount corresponding to 0.1 to 0.5 of the total conventional dose of the first active ingredient per administration.

In another preferred embodiment, the total content of the first active ingredient in said n unit dosage forms corresponds to 0.1 to 0.5 of the total conventional dose of the first active ingredient per administration, wherein n is 2, 3, 4 or 5.

In another preferred embodiment, said n unit dosage forms are the same or different.

In another preferred embodiment, the first active ingredient is present in the unit dosage form in an amount of from 10 to 160mg per dose, preferably from 10 to 100mg per dose, more preferably from 10 to 80mg per dose, such as 10, 20, 30, 40, 50, 60, 70, 80mg per dose.

In another preferred embodiment, said unit dosage form is administered in a dose of 20-160mg/50kg body weight, preferably 30-100mg/50kg body weight, based on said first active ingredient.

In another preferred embodiment, the unit dosage form is a dosage form containing a unit dose of the active ingredient.

In another preferred embodiment, the unit dose refers to a single administered dose of the active ingredient.

In another preferred embodiment, the first active ingredient is an antibody or a small molecule compound.

In another preferred embodiment, the VEGF signaling pathway blocker includes an antibody blocking VEGF signaling pathway and a Tyrosine Kinase Inhibitor (TKI).

In another preferred embodiment, the antibody for blocking VEGF signaling pathway comprises: ramucirumab (DC101/ramucirumab), bevacizumab (Avastin), or a combination thereof.

In another preferred embodiment, the tyrosine kinase inhibitor comprises: sunitinib (Sunitinib), polygiemide (Sorafinib), or a combination thereof.

In another preferred embodiment, the antibody that blocks the VEGF signaling pathway is administered at a dose of 0.1-4mg/kg, preferably 0.2-2.5mg/kg, more preferably 0.5-1 mg/kg.

In another preferred embodiment, the tumor immunotherapeutic agent comprises a CAR-T cell, a CAR-NK cell, a tumor vaccine, and an immune checkpoint inhibitor.

In another preferred embodiment, the CAR-T cells are administered at a dose of 1x10⁷-5x10⁸CAR-T cells/kg.

In another preferred embodiment, the first active ingredient and the second active ingredient are mixed or present separately.

In another preferred embodiment, the total content of the first active ingredient and the second active ingredient is 70 to 100wt%, preferably 80 to 100wt%, and more preferably 90 to 100wt%, based on the total weight of the active ingredients in the pharmaceutical composition.

In another preferred embodiment, the solid tumor is selected from the group consisting of: breast tumor, liver tumor, lung tumor, seminal vesicle tumor, pancreatic cancer, gastric cancer, colorectal cancer, esophageal cancer, brain glioma, or a combination thereof.

In another preferred embodiment, the tumor immunotherapy comprises CAR-T cell therapy, CAR-NK cell therapy, tumor vaccine therapy, immune checkpoint inhibitor therapy, or a combination thereof.

In another preferred embodiment, the immunodetection point inhibitor comprises a PD-1 antibody and a CTLA-4 antibody.

In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In another preferred embodiment, the dosage form of the pharmaceutical composition comprises injection and oral preparation.

In a second aspect of the invention, there is provided the use of a low dose of a VEGF signaling pathway blocker for the preparation of a pharmaceutical composition or kit for enhancing the efficacy of tumor immunotherapy,

wherein, the low dose refers to that the administration dose A1 of the VEGF signal pathway blocker is A1 ≦ 1/2A0, preferably A1 ≦ 1/4A0 compared with the conventional administration dose A0.

In another preferred embodiment, the VEGF signaling pathway blocker comprises an antibody that blocks the VEGF signaling pathway.

In another preferred embodiment, the kit comprises 2-4 unit dosage forms, and the amount of the VEGF signaling pathway blocker, such as an antibody blocking VEGF signaling pathway, in each unit dosage form is 0.1-0.5 (preferably 0.15-0.4, or 0.2-0.25) of the conventional dose, wherein the conventional dose is 200-400mg/50kg body weight.

In another preferred embodiment, the pharmaceutical composition is a unit dosage form, and the amount of the VEGF signaling pathway blocker, such as an antibody for blocking VEGF signaling pathway, in the unit dosage form is 0.1 to 0.5 (preferably 0.15-0.4, or 0.2-0.25) of the conventional dose, wherein the conventional dose is 200-400mg/50kg body weight.

In a third aspect of the invention, there is provided a kit for enhancing the effect of immunotherapy on a tumour, the kit comprising:

(a) a first pharmaceutical composition comprising a VEGF signaling pathway blocker, and a pharmaceutically acceptable carrier, and being a unit dosage form, and the kit comprising 2-4 unit dosage forms, wherein the amount of the VEGF signaling pathway blocker in the unit dosage forms is 0.1 to 0.5 (preferably 0.15-0.4, or 0.2-0.25) of the conventional dose, wherein the conventional dose is 200-400mg/50kg body weight;

(b) optionally a second pharmaceutical composition, said second pharmaceutical composition being a tumor immunotherapeutic agent;

(c) and (6) instructions.

In another preferred embodiment, the second pharmaceutical composition is a cell preparation.

In another preferred embodiment, the second pharmaceutical composition is a CAR-T formulation.

In another preferred embodiment, the CAR-T formulation comprises an autologous or allogeneic CAR-T formulation.

In another preferred embodiment, the first pharmaceutical composition and the second pharmaceutical composition are independent of each other.

In another preferred embodiment, the first and second pharmaceutical compositions are in separate packages or containers.

In another preferred embodiment, the description describes a method of treatment according to the fourth aspect of the invention.

In a fourth aspect of the present invention, there is provided a method of increasing the effectiveness of tumor immunotherapy, comprising the steps of:

(I) administering to a subject in need thereof a low dose of a VEGF signaling pathway blocker;

(II) subjecting the subject to tumor immunotherapy,

wherein, the low dose refers to the administration dosage A1 of the VEGF signal pathway blocker, compared with the conventional administration dosage A0, A1 is less than or equal to 1/2A0, preferably A1 is less than or equal to 1/4A 0.

In another preferred embodiment, step (I) comprises administering to said subject two or three times an antibody that blocks the VEGF signaling pathway, with the interval between successive administrations being t 1.

In another preferred embodiment, t1 is 3-21 days, preferably 4-15 days, and more preferably 7-14 days.

In another preferred embodiment, said step (II) is carried out at a time t2 after the last application in step (I).

In another preferred embodiment, t2 is 3-21 days, preferably 4-15 days, and more preferably 7-14 days.

In another preferred embodiment, the subject includes humans and non-human mammals (e.g., rodents and primates).

In another preferred embodiment, step (II) is followed by the steps of:

(III) administering to the subject a low dose of an antibody that blocks the VEGF signaling pathway.

In another preferred embodiment, said step (III) is carried out with a time interval t3 after the last application in step (II).

In another preferred embodiment, t3 is 3-21 days, preferably 4-15 days, and more preferably 7-14 days.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows that low doses of anti-angiogenic drugs can induce tumor vessel normalization. Wherein, figure 1A shows a representative immunohistochemical staining profile, endothelial cell marker CD31 molecule showing red fluorescence, pericyte marker NG2 molecule showing green fluorescence; FIG. 1B shows statistics of tumor vascular density, and FIG. 1C shows statistics of pericyte coverage.

Figure 2 shows that low dose anti-angiogenic drug treatment can enhance the cancer suppressing effect of tumor vaccines. In the figure, D13, D16, D19 and D22 respectively represent

days

13, 16, 19 and 22 after the first inoculation, and D10 represents the injection of 10mg/kg of the anti-angiogenic drug DC 101.

Figure 3 shows that low dose anti-angiogenic drug treatment can promote T cell tumor infiltration. Wherein, fig. 3A shows tumor infiltration of CD4+ T cells; fig. 3B shows tumor infiltration of CD8+ T cells.

Figure 4 shows the effect of conventional doses of anti-angiogenic drugs on tumor vaccine therapy. In the figure, D7, D10, D13 and D16 respectively represent

days

7, 10, 13 and 16 after the first inoculation, and D40 represents that 40mg/kg of anti-angiogenesis drug DC101 is injected.

Figure 5 shows the results of pathological section analysis of CAR-T cell interventional therapy in tumor patients pretreated with low dose drugs blocking the VEGF signaling pathway.

Detailed Description

The present inventors have conducted extensive and intensive studies and, for the first time, have unexpectedly found that a low dose of a VEGF signaling pathway blocker, such as an antibody that blocks VEGF signaling pathway, can significantly improve the effect of tumor immunotherapy and promote infiltration of T lymphocytes into tumor tissues. Experiments show that CAR-T cells are injected into solid tumor tissues by using an intervention strategy, and meanwhile, the CAR-T cells are matched with a low-dose antibody for blocking a VEGF signal pathway to induce the normalization of tumor blood vessels, so that tumors at the injection part have obvious necrosis. On the basis of this, the present invention has been completed.

VEGF

Vascular Endothelial Growth Factor (VEGF), also known as Vascular Permeability Factor (VPF) in the early days, is a heparin-binding growth factor (vascular endothelial cell-specific) that induces angiogenesis in vivo.

Studies have shown that the volume of solid tumors exceeds 2-3mm³In time, the growth of tumor bodies cannot be supported by pure oxygen diffusion, and the growth of tumor bodies can be supported by the formation of new blood vessels. Compared with normal blood vessels, the tumor neovascularization has the phenomena of vasodilatation, tortuosity and saccular structure formation, generates irregularly connected branches, and has uneven blood vessel density distribution; the loss of the caliber adjusting mechanism leads to abnormal shunt, and the vascular permeability and the vascular clearance are both obviously increased. Abnormal tumor neovascularization can finally cause uneven blood vessel distribution, blood flow stagnation in partial areas, aggravate the formation of tumor hypoxia microenvironment, further influence the tumor gene phenotype, activate angiogenesis factors including Vascular Endothelial Growth Factor (VEGF), promote the generation of tumor angiogenesis, cause the instability of tumor cell genes, activate certain tumor cell survival factors, cause the tolerance of tumor cells to radiotherapy and chemotherapy, and further promote tumor metastasis.

VEGF signaling pathway blockers

As used herein, a "VEGF signaling pathway blocker" refers to inhibition of the vascular regulatory function of VEGF by blocking its signaling pathway. VEGF signaling pathway blockers include antibodies and Tyrosine Kinase Inhibitors (TKIs) that block the VEGF signaling pathway.

"antibody blocking VEGF signaling pathway" refers to a receptor or its ligand that specifically binds to vascular endothelial growth factor, a typical anti-angiogenic drug.

There are 2 clinically used antibody drugs blocking VEGF signaling pathway: ramucirumab (Ramucirumab) and bevacizumab (Avastin).

Ramucirumab (Ramucirumab) is a fully human monoclonal antibody, and its murine monoclonal antibody is DC 101. It specifically binds to vascular endothelial growth factor receptor 2(VEGFR2/KDR) and thus prevents receptor activation, so it is an inhibitor of VEGFR 2. Ramucirumab has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of some solid tumors, including gastric cancer and metastatic non-small cell lung cancer (NSCLC).

Bevacizumab (Avastin) is a recombinant humanized monoclonal IgG1 antibody that acts by binding VEGF ligands to block its binding to endothelial cell surface receptors (Flt-1 and KDR), thereby inhibiting the biological activity of vascular endothelial growth factor. Bevacizumab has been approved by the U.S. food and drug administration for the treatment of a range of cancers, including colon, lung, kidney, and brain cancers, among others.

Clinically, the conventional dose of Ramucirumab is 8 mg/kg; a conventional dose of Avastin is 5 mg/kg. Based on the results of the mouse experiments in the examples, applicants believe that 1/5-1/10 are preferred doses, but do not exclude different dose ranges at different tumors and different stages of tumors and in combination with different tumor immunotherapies.

Compound pharmaceutical composition and medicine box

The present invention provides a composition comprising a first active ingredient a VEGF signaling pathway blocker, such as an antibody that blocks the VEGF signaling pathway, and a second active ingredient a tumor immunotherapeutic agent; and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The pharmaceutical combination of the present invention may also be formulated as a powder for inhalation by nebulization. One preferred dosage form is an injectable formulation. In addition, the pharmaceutical compositions of the present invention may also be used with other therapeutic agents.

The present invention also provides a kit for enhancing the effect of immunotherapy on tumors, comprising:

(a) a first pharmaceutical composition comprising a VEGF signaling pathway blocker, preferably an antibody that blocks VEGF signaling pathway, and a pharmaceutically acceptable carrier, and being a unit dosage form, and the kit comprising 2-4 unit dosage forms, wherein the VEGF signaling pathway blocker is contained in the unit dosage form in an amount of 0.1 to 0.5 (preferably 0.15-0.4, or 0.2-0.25) of a conventional dose, wherein the conventional dose is 400mg/50kg body weight;

(c) and (6) instructions.

The pharmaceutical composition or kit of the invention is suitable for the treatment of tumors, in particular for the treatment of solid tumors.

It is noted that the pharmaceutical composition or kit of the present invention contains a low dose of a VEGF signaling pathway blocker, such as an antibody blocking VEGF signaling pathway, wherein the low dose means that the administered dose of the VEGF signaling pathway blocking antibody a1 is a1 ≦ 1/2a0, preferably a1 ≦ 1/4a0, as compared to the conventional administered dose of a 0.

In another preferred embodiment, the pharmaceutical composition of the invention is in unit dosage form, the active ingredient being present in each unit dosage form in an amount of 0.1 to 1 (or 0.25-1, or 0.5-1) per daily dose, wherein the daily dose of the first active ingredient is 20-100mg (for a 50kg human).

Of course, the effective dose of the active ingredient used may vary depending on the mode of administration and the severity of the disease to be treated, etc.

Method of treatment

The invention also provides a method for treating solid tumors with two active ingredients of the invention or corresponding medicaments, which comprises administering to a mammal a low dose of an antibody blocking the VEGF signaling pathway of the first active ingredient and a tumor immunotherapeutic agent of the second active ingredient, or a pharmaceutical composition comprising the first active ingredient and the second active ingredient.

When both active ingredients of the present invention are used for the above purposes, they may be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, and the like, and may be administered parenterally in the form of sterile injectable solutions or suspensions (containing about 0.05-5% suspending agent in an isotonic medium). For example, these pharmaceutical preparations may contain from about 0.01% to about 99%, more preferably from about 0.1% to about 90%, by weight of the active ingredient in admixture with a carrier.

The two active ingredients or pharmaceutical compositions of the present invention may be administered by conventional routes including, but not limited to: intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral, or topical administration. Preferred routes of administration include intratumoral or intravenous administration.

From the standpoint of ease of administration, the preferred pharmaceutical composition is a liquid composition.

In addition, the two active ingredients or drugs of the present invention can also be used in combination with other drugs for treating cancer (such as cisplatin, paclitaxel, etc.).

The main advantages of the invention include:

(a) low doses of VEGF signaling pathway blockers, such as antibodies that block VEGF signaling pathways, can significantly improve the effectiveness of tumor immunotherapy.

(b) Low doses of VEGF signaling pathway blockers, such as antibodies that block the VEGF signaling pathway, can promote infiltration of T lymphocytes into tumor tissue.

(c) Low doses of VEGF signaling pathway blockers, such as antibodies that block the VEGF signaling pathway, may also promote the effects of other methods of tumor treatment, such as chemotherapy and radiation therapy.

(d) The medicine composition is suitable for treating solid tumors.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Universal material

In the examples below, the VEGF signaling pathway blocking drug DC101 was purchased from Bio X Cell, and rabbit anti-mouse IgG monoclonal antibody used as a control was purchased from Jackson ImmunoResearch. An MCaP0008 tumor cell vaccine, mitomycin-treated MCaP0008 tumor cells, induces an immune response specific for the MCaP0008 tumor antigen by antigen presenting cells in vivo. The anti-angiogenic drug bevacizumab is provided by hospitals; CAR-T cells for cellular immunotherapy are produced by Boshengji.

Example 1

Effect of Low dose of antiangiogenic drugs on tumor vasculature

An in situ MCaP0008 breast cancer model was constructed and when the tumor diameter of MCaP0008 breast cancer mice reached 4-5mm, the mice were given a first low dose (1/4 for a mouse universal dose) of the VEGF signaling pathway blocking drug (DC101) and treatment with rabbit anti-mouse IgG mab (control) (set to 0 days) at a drug injection frequency of 10mg/kg (1/4 for a mouse universal dose only) once every three days for a total of 4 administrations. On day 11, mice were perfused with 4% paraformaldehyde, tumor tissue cryosections (20 μm) were prepared, and the tissue sections were subjected to marker staining for the endothelial marker CD31 molecule and the pericyte marker NG2 molecule. Tumor regions were randomly selected by confocal laser scanning microscopy for immunohistochemical image acquisition (4-6 regions per tumor tissue, 6-8 tumors per group) at 20 x magnification. And (4) carrying out statistical analysis on tumor vascular density and pericyte coverage rate.

Results as shown in fig. 1, a significant decrease in tumor vessel density (fig. 1A and 1B), and a significant increase in pericyte coverage (fig. 1A and 1C) were still observed at day 11 after low dose anti-angiogenic drug DC101(10mg/kg) treatment. The results indicate that low doses of anti-angiogenic drugs can induce long-lasting tumor vessel normalization.

Example 2

Effect of Low dose anti-angiogenic drug combination vaccine treatment on mouse tumors

In order to study the effect of tumor vessel normalization on tumor immunotherapy, low dose anti-angiogenic drug combination vaccine therapy experiments were performed. When the tumor diameter of the breast cancer mouse tumor model MCaP0008 reaches 3mm, the tumor is randomly grouped and injected into the abdominal cavity by 5 multiplied by 10⁶Mitomycin-pretreated MCaP0008 tumor cell vaccine or PBS (control) with equal volume, and the injection time is 7, 9, 12 and 14 days respectively. Anti-angiogenic drugs DC101(10mg/kg) or rabbit anti-mouse IgG (10mg/kg) were injected on

days

13, 16, 19, and 20, respectively. Tumor size was measured every three days starting on day 13. And (5) drawing a tumor growth curve. Each group contained 10 mice.

The results are shown in fig. 2, the inhibition effect of the vaccine treatment alone on the tumor growth of the mice is not obvious, and the normalization of tumor blood vessels induced by the low dose of the anti-angiogenesis drug can obviously enhance the tumor immunotherapy effect. The results show that low dose anti-angiogenic drug combination vaccine treatment can significantly inhibit mouse tumor growth compared to vaccine treatment alone and low dose anti-angiogenic drug treatment.

Example 3

Effect of Low dose anti-angiogenic drug treatment on tumor infiltration of T cells

To investigate the mechanism by which normalization of tumor blood vessels enhances immunotherapy, CD8+ and CD4+ T lymphocytes infiltrating in tumor tissues were analyzed. When the tumor diameter of MCaP0008 breast cancer mice reaches 4-5mm, the mice are treated by low dose of DC101 and tumor cell vaccine, and the injection frequency of the DC101 and the control rabbit anti-mouse IgG is once every three days, the dose is 10mg/kg, and the administration is carried out for 4 times. Tumor tissues were then collected, single cell suspensions were prepared, and tumor infiltrating CD4+ T cells and CD8+ T cells were analyzed by a flow cytometric analyzer.

The results are shown in FIG. 3, the infiltration capacity of CD8+ and CD4+ lymphocytes can be significantly improved by the single low dose of the VEGF signaling pathway blocking drug (group: PBS/D10), while the infiltration capacity of CD4+ lymphocytes is further significantly enhanced after the low dose of the VEGF signaling pathway blocking drug combined vaccine treatment (group: vaccine/D10). The results indicate that low dose anti-angiogenic drug treatment can promote T cell tumor infiltration.

Example 4

Effect of conventional doses of antiangiogenic drugs on tumor vaccine therapy

The experimental conditions were the same as in example 2, except that the dose of the anti-angiogenic drug administered was 40mg/kg, i.e., a conventional dose of the anti-angiogenic drug was administered, and the effect on the tumor immunotherapy was investigated.

The results are shown in fig. 4, where the effect of the vaccine alone on the inhibition of tumor growth in mice was not significant. Meanwhile, the inhibition effect of the combination treatment of the anti-angiogenesis drug with the conventional dose and the vaccine on the tumor growth of the mice is not obviously different from the treatment effect of the anti-angiogenesis drug with the single conventional dose. The above results indicate that conventional (high) doses of anti-angiogenic drugs do not improve the therapeutic efficacy of tumor vaccines.

Example 5

CAR-T clinical treatment combining angionormalization pretreatment with interventional therapy

CAR-T cell therapy is a promising new cellular immunotherapy that can effectively treat tumors. Patients with relapsed or refractory acute lymphoblastic leukemia receiving CD19CAR-T therapy had a complete remission rate of 90%. However, CAR-T cells do not perform satisfactorily in the treatment of solid tumors, and international attempts at CAR-T cell therapy for solid tumors have not been successful. Factors such as uneven blood vessel distribution inside tumor tissues and insufficient oxygen supply inside the tumor seriously hinder the curative effectiveness of a CAR-T cell system reinfusion scheme on solid tumors, so that the CAR-T cells which are systemically reinfused cannot be completely and effectively infiltrated into the tumor tissues, and the CAR-T cells cannot be completely and effectively activated, proliferated and retained inside the tumor for a long time. Therefore, in order to ameliorate the adverse effects of the specificity of the above-mentioned solid-triomas on CAR-T cell therapy, intervention strategies were chosen to inject CAR-T cells inside tumor tissue while inducing tumor vessel normalization in conjunction with low doses (1/5 and 1/10 normal doses) of Avastin (bevacizumab, anti-angiogenic drugs).

Clinical scientific trials were conducted on 1 patient with MUC1+ advanced solid tumors (advanced seminal vesicle adenocarcinoma) who signed an informed consent, after approval by the ethical committee of clinical hospitals. The patient is firstly injected with 1mg/kg (normal dose of 1/5, the dose is obtained by conjecture according to the test result of the mouse) of the VEGF signal pathway blocking drug Avastin (bevacizumab, targeting ligand VEGF), the same dose of VEGF signal pathway blocking drug is repeatedly injected after 2 weeks, and then the intratumoral injection of MUC1CAR-T cells is carried out on the metastatic tumor focus of the patient under the guidance of color Doppler ultrasound after 1 week. 1 week after CAR-T cell injection, the VEGF signaling pathway blocking drug was injected again, but the dose was reduced to 0.5mg/kg (1/10, the normal dose, which was presumed from the above mouse assay results). After 3 weeks of CAR-T cell injection, tumor tissues were surgically harvested and made into paraffin sections for HE staining.

The results are shown in FIG. 5, where the pink oval tissue is freshly necrosed tumor cells, and the cracks formed by the necrosed tumor cells and the surrounding tissue can be seen. The result shows that the tumor bleeding tumor injected by the low-dose VEGF signal pathway blocking drug and the MUC1CAR-T combined CAR-T cell has obvious necrosis and shows good treatment effect.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A pharmaceutical composition for treating solid tumors, comprising a first active ingredient which is a VEGF signaling pathway blocker and a second active ingredient which is a tumor immunotherapeutic agent,

and the pharmaceutical composition is in unit dosage form, the content of the first active ingredient in each unit dosage form is 10-160 mg/dose,

and the VEGF signal pathway blocker is an antibody for blocking a VEGF signal pathway, and the antibody is ramucirumab or bevacizumab.

2. The pharmaceutical composition of claim 1, wherein the first active ingredient is present in an amount corresponding to 0.1 to 0.5 of the total conventional dose of the first active ingredient per administration in one unit dosage form.

3. The pharmaceutical composition of claim 1, wherein the first active ingredient is present in an amount of 10 to 100mg per dose per unit dosage form.

4. The pharmaceutical composition of claim 1, wherein the unit dosage form is a dosage form comprising a unit dose of the active ingredient, and wherein the unit dose is a single administered dose of the active ingredient.

5. The pharmaceutical composition of claim 1, wherein the first active ingredient and the second active ingredient are mixed or present separately.

6. The pharmaceutical composition according to claim 1, wherein the total content of the first active ingredient and the second active ingredient is 70 to 100wt% based on the total weight of the active ingredients in the pharmaceutical composition.

7. The pharmaceutical composition of claim 1, wherein the solid tumor is selected from the group consisting of: breast tumor, liver tumor, lung tumor, seminal vesicle tumor, pancreatic cancer, gastric cancer, colorectal cancer, esophageal cancer, brain glioma, or a combination thereof.

8. The pharmaceutical composition of claim 1, wherein the tumor immunotherapeutic agent comprises a CAR-T cell, a CAR-NK cell, a tumor vaccine, and an immune checkpoint inhibitor.

9. The pharmaceutical composition of claim 8, wherein the immune checkpoint inhibitor comprises a PD-1 antibody and a CTLA-4 antibody.

10. The pharmaceutical composition of claim 1, wherein the dosage form of the pharmaceutical composition comprises an injection and an oral preparation.

11. Use of a low dose of a VEGF signaling pathway blocker in the preparation of a pharmaceutical composition or kit for enhancing the efficacy of tumor immunotherapy,

wherein, the low dose refers to that A1 is less than or equal to 1/2A0 when the administration dose A1 of the VEGF signal pathway blocker is compared with the conventional administration dose A0;

wherein the VEGF signal pathway blocker is an antibody for blocking a VEGF signal pathway, the antibody is ramucirumab or bevacizumab,

and the kit comprises 2-4 unit dosage forms, and the content of the antibody of the VEGF signal pathway blocker in each unit dosage form is 10-160 mg/dose.

12. A kit for enhancing the immunotherapeutic effect of a tumor, the kit comprising:

(a) a first pharmaceutical composition comprising a VEGF signaling pathway blocker, and a pharmaceutically acceptable carrier, and being in unit dosage form, and the kit comprising 2-4 unit dosage forms, wherein the amount of VEGF signaling pathway blocker in the unit dosage forms is 10-160 mg/dose,

the VEGF signal pathway blocker is an antibody for blocking a VEGF signal pathway, and the antibody is ramucirumab or bevacizumab;

(c) and (6) instructions.