CN111000859A

CN111000859A - Methods and kits for treating cancer by combination therapy

Info

Publication number: CN111000859A
Application number: CN201910951479.XA
Authority: CN
Inventors: 梁丽娴; 姜志宏; 刘良; 萧文鸾; 黄居敏
Original assignee: Macau Univ of Science and Technology
Current assignee: Macau Univ of Science and Technology
Priority date: 2018-10-05
Filing date: 2019-10-08
Publication date: 2020-04-14
Also published as: AU2018101488A4

Abstract

A method of treating a subject having cancer, the method comprising administering to the subject an effective amount of a polysaccharide extract and an effective amount of a PD-1 inhibitor, wherein the polysaccharide extract comprises ginseng polysaccharide. A method of increasing the number of tumor infiltrating lymphocytes in cancer cells of a subject; and a pharmaceutical combination comprising the polysaccharide extract and the PD-1 inhibitor.

Description

Methods and kits for treating cancer by combination therapy

Technical Field

The present invention relates to a method of treating cancer, in particular, but not exclusively, by combination therapy. The invention also relates to pharmaceutical combinations for use in said method.

Background

Immunotherapy enhances the immune system of patients to combat cancer and has become a promising source of new treatments for cancer. Among the numerous immunotherapeutic strategies, immune checkpoint blockers show significant advantages in the treatment of various cancers. Immune checkpoint blockers enhance anti-tumor immunity by blocking intrinsic immune down-regulating factors, such as cytotoxic T lymphocyte antigen 4(CTLA-4) and programmed cell death protein 1(PD-1) or its ligand (programmed cell death ligand 1(PD-L1) 2). These immune checkpoint inhibitors exert their effects by disrupting the interaction between the inhibitory receptor PD-1 and its ligand PD-L1, causing restoration of antitumor lymphocyte function. Several antibodies against immune checkpoints cause patients with a variety of cancers to mount a durable clinical response and have acquired U.S. Food and Drug Administration (FDA) approval (Garon, e.b., et al, Pembrolizumab for the treatment of non-small-cell lung cancer. nengl J Med,2015.372(21): p.2018-28). However, 85% of unselected patients do not respond to treatment and almost all initially responding patients eventually develop a drug-resistant disease. In addition, immune checkpoint blockers may produce immune-related side effects (Oncololist. 2016Oct; 21(10): 1230-.

Currently, several combination therapies have been applied for cancer treatment. For example, for patients with diseases resistant to PD-1 blockers, the use of combination therapy offers the possibility of targeting additional pathways, thereby increasing the proportion of patients who achieve a response. The IL-15 superagonist ALT-803 is used in combination with nivolumab (nivolumab) to improve survival in patients with metastatic non-small cell lung cancer. The overall survival of nivolumab plus nivolumab in combination therapy or nivolumab alone was significantly longer in patients with advanced melanoma than for nivolumab alone. However, in view of the frequent existence of resistance mechanisms, there is still a need for therapeutic approaches for the treatment of cancer, especially those that have fewer adverse effects and suitably inhibit PD-1.

Disclosure of Invention

In a first aspect, the invention provides a method of treating a subject having cancer, the method comprising administering to the subject an effective amount of a polysaccharide extract and an effective amount of a PD-1 inhibitor, wherein the polysaccharide extract comprises ginseng polysaccharide.

In a second aspect, the invention relates to a method of increasing the number of tumor-infiltrating lymphocytes in cancer cells of a subject, comprising administering to the subject an effective amount of a polysaccharide extract and an effective amount of a PD-1 inhibitor, wherein the polysaccharide extract comprises ginseng polysaccharide.

In a third aspect, the present invention relates to a pharmaceutical combination comprising a polysaccharide extract and a PD-1 inhibitor, wherein the polysaccharide extract comprises ginseng polysaccharides.

The inventors have surprisingly found that the combination of the polysaccharide extract of the invention and a PD-1 inhibitor is effective in inhibiting the growth and development of tumors, including cancer. The combination can be used for treating diseases caused by macrophage (especially CD 4)⁺T and CD8⁺T lymphocytes) infiltration of the tumor or cancerous tissue, further enhancing the immune system of the subject. Accordingly, the inventors have developed a new method for treating cancer or alleviating symptoms associated with cancer by enhancing the immune system or delaying the progression of the disease. The invention is particularly useful for treating lung cancer (e.g., non-small cell lung cancer) and reducing the adverse side effects of chemotherapeutic compounds.

Further features and aspects of the invention will become apparent by reference to the following detailed description and drawings.

Drawings

FIG. 1 is a graph showing tumor size after treatment of an animal model in one embodiment of the invention with a combination of polysaccharide extract (labeled GP) and anti-PD-1 antibody (labeled anti-PD-1), polysaccharide extract alone, anti-PD-1 antibody alone, or Phosphate Buffered Saline (PBS) as a control (compared to the anti-PD-1 antibody treated group), respectively^*P<0.05 and^**P<0.01; mean ± SEM).

Figure 2 is a photograph showing the size of the tumor of the animal model of figure 1 after treatment.

FIG. 3 is a graph of the post-treatment weight of the tumors of FIG. 1, data expressed as mean. + -. SEM, compared to anti-PD-1 antibody treated groups^*P<0.05。

Figure 4 is a graph showing body weight of an animal model during treatment in an example of the invention.

FIG. 5 is a microscopic image comparison of excised tumor tissue obtained from animal models following treatment with anti-PD-1 antibody, polysaccharide extract (GP), or a combination of anti-PD-1 antibody and polysaccharide extract to PBS treated controls. These images show the expression of CD4 and CD8 in tissues. Pixel, 400 x.

Detailed Description

Unless defined otherwise, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, "comprising" means including the following elements but not excluding others. By "consisting essentially of …, it is meant that the material consists of the elements as well as common and unavoidable impurities (e.g., by-products) and components that typically result from the respective preparation or process used to obtain the material, such as additional trace amounts of components or solvents. By "consisting of …" is meant that the material consists only of …, i.e., is formed from the elements. As used herein, the terms "a", "an" and "the" are intended to include both the singular and the plural, unless the context clearly indicates otherwise.

In a first aspect, the present invention provides a method of treating a tumour, particularly a cancer, by combination therapy. The method comprises the step of administering to the subject an effective amount of the polysaccharide extract and an effective amount of a PD-1 inhibitor.

The term "polysaccharide extract" refers to a mixture obtained from a substance such as an animal, plant or fungus, the polysaccharide being present in the mixture in an amount of at least 50% by weight of the total weight of the mixture. The polysaccharide content of the polysaccharide extract is at least 50%, 60%, 70%, 80% or 90% by weight of the total weight of the polysaccharide extract. Preferably, the polysaccharide extract comprises neutral polysaccharides, acidic polysaccharides, or a combination thereof; or the polysaccharide consists essentially of a neutral polysaccharide, an acidic polysaccharide, or a combination thereof.

In one embodiment, the polysaccharide in the polysaccharide extract has a molecular weight of 50,000 to 1,000,000, 80,000 to 750,000, or 100,000 to 500,000. Polysaccharides having an average molecular size within any of these ranges are suitable for use in combination with a PD-1 inhibitor to inhibit tumor growth and proliferation.

The polysaccharide extract of the present invention is preferably obtained by extraction of at least one plant material, such as but not limited to a plant-based traditional Chinese medicine. Preferably, the polysaccharide extract comprises ginseng polysaccharide. The ginseng polysaccharide is extracted from ginseng. Ginseng refers to the root part of any plant in the genus Panax (Panax), such as Panax ginseng (Panax ginseng), Panax notoginseng (Panax notoginseng), Panax quinquefolium (Panax quinquefolius), etc., and any root tuber part of the genus. In one embodiment, the polysaccharide extract comprises ginseng polysaccharides from ginseng (Panax ginseng).

In another embodiment, the polysaccharide extract further comprises a polysaccharide extracted from at least one of dendrobium (Dendrobii Caulis), ginseng (Panax ginseng), Astragalus (Radix Astragali) and Poria (Poria), in other words, the polysaccharide extract is prepared by extracting and separating polysaccharide components from ginseng (Panax ginseng), dendrobium, Astragalus and Poria. In particular, the molecular weight of the polysaccharide is 100,000-500,000.

Preferably, the polysaccharide extract is obtained by performing the following steps:

-providing the above herbal materials in a solvent (e.g. water or alcohol);

-heating the herbal material for 1-5 hours, 2-4 hours, or 3 hours, once to twice;

-the boiled mixture is either left overnight at room temperature or centrifuged at 3000g for 30 minutes;

-collecting the supernatant and removing the solvent from the supernatant;

precipitating the supernatant with alcohol, in particular anhydrous alcohol, and

-separating the resulting mixture by column chromatography.

In one embodiment of performing the above steps, 200-1000 g of Ginseng, 200-1000 g of dendrobium, 200-1000 g of Ginseng (Panax Ginseng), 200-1000 g of astragalus or 200-1000 g of poria is immersed in a solvent and heated (especially boiled once) for about 1-5 hours, 2-4 hours or 3 hours to extract.

Preferably, the column chromatography is performed using a DEAS-cellulose column. The separated fractions include a neutral polysaccharide fraction and an acidic polysaccharide fraction. More preferably, these fractions are subsequently subjected to size exclusion column chromatography to obtain polysaccharides having a molecular weight in the range of 50,000-1,000,000, 80,000-750,000, or 100,000-500,000, preferably polysaccharides having a molecular weight in the range of 100,000-500,000 MW.

The PD-1 inhibitor (programmed cell death protein-1 inhibitor) of the present invention refers to any substance capable of targeting PD-1 present on the cell surface. Without being limited by theory, a blocker of PD-1 may activate the immune system of a cell or subject to combat cancer. Preferably, the PD-1 inhibitor is an anti-PD-1 antibody, especially an anti-PD-1 monoclonal antibody. In one embodiment, the anti-PD-1 antibody is a commercially available (e.g., but not exclusively, from Bio X cell) cloning site, RMP1-14, catalog # BE 0146.

Unexpectedly, the inventors found that the polysaccharide extract of the present invention works well in combination with PD-1 inhibitors, enhancing the anti-tumor effect. The combination of polysaccharide extract with PD-1 inhibitor significantly inhibited the growth and development of cancer cells in vivo and enhanced the immune system of mice by promoting the recruitment and activation of tumor infiltrating lymphocytes.

The expressions "effective amount" and "effective dose" generally refer to an amount sufficient to produce the desired result of treatment, wherein the exact nature of the result will vary depending on the particular disease being treated. In the present invention, the disease is cancer, and thus the result is generally inhibition or inhibition of cancer cell proliferation, reduction of cancer cells or improvement of symptoms associated with cancer cells. Especially the size of the tumor will shrink after treatment.

Effective amounts of the polysaccharide extract and PD-1 inhibitor of the invention may depend on the species, weight, age and individual condition of the subject and may be determined by standard procedures, e.g., with cell cultures or experimental animals. In one embodiment, the dose of the polysaccharide extract of the invention for treating a subject may be, for example, 100mg/kg, 150mg/kg, 200mg/kg or 250 mg/kg. In another embodiment, the dose of the polysaccharide extract of the invention for treating a subject may be, for example, about 5mg/kg to 25mg/kg, 8mg/kg to 20mg/kg, or 10mg/kg to 15mg/kg when the subject is a human. Further, the dose of the PD-1 inhibitor for treating a subject may be 5mg/kg to 20mg/kg, 10mg/kg to 15mg/kg, 10mg/kg, or 15 mg/kg. Alternatively, the dose of the PD-1 inhibitor may be 0.3mg/kg to 2mg/kg, 0.5mg/kg to 1mg/kg, or 0.8mg/kg to 1mg/kg when the subject is a human.

The subject may be a human or an animal, in particular a mammal, preferably a human.

Both the polysaccharide extract and the PD-1 inhibitor may be provided in a pharmaceutical composition comprising a physiologically tolerable excipient. The skilled person is able to select suitable excipients depending on the form of the pharmaceutical composition and knows the method of manufacturing the pharmaceutical composition and is able to select suitable methods to prepare the pharmaceutical composition depending on the type of excipient and the form of the pharmaceutical composition. In particular, the polysaccharide extract and the PD-1 inhibitor according to the invention may each be present in solid, semi-solid or liquid form for administration to a subject (preferably to a human) via oral, rectal, topical, parenteral, or transdermal or inhalation routes. Preferably, the polysaccharide extract is formulated in a liquid (e.g., suspension or solution) form for administration to a subject by the oral route. Preferably, the PD-1 inhibitor is configured in liquid form for administration to a subject by injection (such as, but not limited to, intravenous, intraperitoneal, intramuscular, subcutaneous, and intradermal routes).

The polysaccharide extract may be administered before, after or simultaneously with the PD-1 inhibitor during administration. In one embodiment, the polysaccharide extract is administered to the subject orally daily during the course of administration, and the PD-1 inhibitor is administered to the subject continuously by injection every 1 day, 2 days, 3 days, 4 days, or 5 days. Preferably, the PD-1 inhibitor is administered to the subject by injection every 3 days. The inventors have found that the continuous administration of the PD-1 inhibitor and the daily administration of the polysaccharide extract improves the immune system of the subject. Without being limited by theory, the polysaccharide extract and PD-1 inhibitor promote infiltration of lymphocytes into tumors and cause apoptosis of cancer cells. Combination therapy can help reduce adverse effects and reduce the progression of drug resistance due to chronic administration of PD-1 inhibitors.

The term "cancer" refers to or describes a physiological condition of a subject in which a population of cells is characterized by uncontrolled cell growth. The term "tumor" refers to merely a benign (generally harmless) or malignant (cancerous) growing mass. In one embodiment, preferably, the cancer is selected from lung cancer, breast cancer, colorectal cancer or renal cancer. Preferably, the cancer is lung cancer, especially non-small cell lung cancer.

The invention also relates to a method of increasing the number of tumor infiltrating lymphocytes in a cancer cell or tumor in a subject. The method comprises the step of administering to the subject an effective amount of the polysaccharide extract and an effective amount of a PD-1 inhibitor. The polysaccharide extract and PD-1 inhibitor are as described above.

By "tumor infiltrating lymphocyte" is meant any lymphocyte, particularly a T lymphocyte, that is capable of recognizing and targeting a transformed cell prior to the cell forming a tumor, thereby eliminating the cell. An increase in the number of tumor-infiltrating lymphocytes in a cancer cell or tumor generally indicates an increased recruitment and activation of tumor-infiltrating lymphocytes in the cancer cell or tumor, resulting in a reduction in the size of the cancer cell or tumor.

In one embodiment, the tumor infiltrating lymphocyte is a T lymphocyte, preferably selected from the group consisting of CD8⁺T lymphocytes, CD4⁺T lymphocytes, or a combination thereof. In particular, the tumor infiltrating lymphocytes comprise CD8⁺T lymphocytes and CD4⁺T lymphocytes.

In a preferred embodiment, the polysaccharide extract comprises Ginseng polysaccharides, preferably polysaccharides from dendrobe, Ginseng (Panax Ginseng), astragalus and poria. The PD-1 inhibitor is an anti-PD-1 antibody. In particular, the polysaccharide extract is administered to the subject daily by the oral route, while the PD-1 inhibitor is administered to the subject by injection every 1 day, 2 days, 3 days, 4 days or 5 days, or preferably every 3 days.

The inventors have demonstrated that the combination therapy according to the invention is effective in increasing the number of tumor-infiltrating lymphocytes in a tumor of a subject, and that the combination therapy can therefore be used to improve the anti-tumor immune system of a subject.

Furthermore, the present invention relates to a pharmaceutical combination therapy suitable for the above method. The pharmaceutical kit comprises the above polysaccharide extract and a PD-1 inhibitor. The polysaccharide extract comprises ginseng polysaccharides having a molecular weight of preferably 10,000 to 50,000.

Preferably, the polysaccharide extract comprises polysaccharides from dendrobe, Ginseng (Panax Ginseng), astragalus, poria.

In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody.

Accordingly, the present invention provides effective methods for treating cancer and pharmaceutical combinations for such treatment. The invention also relates to application of the polysaccharide extract and the PD-1 inhibitor in preparing a medicament for treating cancer.

In order to better understand the core aspects of the invention, the invention will be further described below by way of examples. The following examples are not intended to limit the scope of protection but are for illustrative purposes.

Example 1

Preparation of polysaccharide extract and PD-1 inhibitor

Grinding traditional Chinese medicines including herba Dendrobii (Dendrobii Caulis), Ginseng radix (Panax Gingseng), radix astragali, and Poria, soaking in deionized water, and extracting with boiling water. In particular, 200g of various traditional Chinese medicines are refluxed for 2 hours by 8-10 times of water (1600-. The extraction is then repeated. After extraction, the mixture was left at room temperature overnight, or the mixture was centrifuged below 3000g for 30 min. The supernatant was then collected and the precipitate was concentrated with 100% ethanol in a volume ratio of 1: 4. The residue was further treated with Sevag reagent to remove proteins, and the supernatant fraction was loaded on a DEAE-cellulose column. The neutral polysaccharide fraction was eluted with an aqueous solvent, while the acidic polysaccharide fraction was eluted with 0.5M sodium chloride. Next, these fractions are concentrated, dialyzed and lyophilized for storage and further use. Separating each polysaccharide fraction on a size exclusion column to obtain polysaccharides having a molecular weight in the range of 100,000-500,000 MW. Subsequently, the saccharide component of the polysaccharide was detected using acid hydrolysis and High Performance Liquid Chromatography (HPLC) analysis. All polysaccharide fractions are combined to form a polysaccharide extract for subsequent use.

The PD-1 inhibitor used was a Bio X cell anti-PD 1 monoclonal antibody (cloning point: RMP1-14, catalog number: BE 0146).

Example 2

Anti-tumor effect on tumor growth of animal model

Animal models, particularly LLC tumor models, were established to evaluate the anti-tumor effect of the combination therapies of the invention (i.e., polysaccharide extracts and PD-1 inhibitors). Animal studies were conducted according to institutional guidelines. 8-10 weeks old C57BL/6 mice were injected with LLC cells subcutaneously (2X 10)⁶) To construct an animal model, namely C57BL/6 mouse Lewis lung cancer model (LLC). On day 7, mice bearing LLC tumors of similar size were randomized into four groups (n-4) treated with control (i.e., PBS), anti-PD-1 antibody (10mg/kg) (RMP1-14, Bio X Cell, 250 μ g per injection), the polysaccharide extract of example 1 (200mg/kg), or a combination of anti-PD-1 antibody and polysaccharide extract. Specifically, anti-PD-1 antibody was injected intraperitoneally every 3 days (i.e., on days 3, 6, 9, 12, 15). The polysaccharide extract was administered by gavage to mice daily. Tumor size and survival were measured as described above. Mice with tumor major diameter sizes greater than 20mm were euthanized for ethical reasons.

Tumor volume (mm)³) Long (mm) x wide (mm)²×/2。

Referring to figures 1 through 3, administration of the combination therapy is effective to inhibit tumor growth in tumor-bearing mice. The combination of polysaccharide extract (GP) and anti-PD-1 antibody resulted in a significant reduction in tumor growth compared to the effects of other therapeutic approaches. Referring to fig. 4, there was no significant change in body weight during the treatment, and no toxic side effects were observed.

Example 3

For CD4⁺And CD8⁺Effect of T lymphocyte Activity

The inventors further evaluated whether the combination therapy of the present invention enhanced CD8⁺And CD4⁺Recruitment and activation of T lymphocyte infiltration. Mice from each treatment group and control group were sacrificed on day 18 and tumor tissues were collected and fixed with 10% formalin. After fixation, the tissue was embedded in paraffin to prepare a slide glass with a tissue section (5 μm). The slices were dewaxed in xylene and dehydrated by a series of alcohol fractionation. Then, it was stained with Mayer's hematoxylin (Sigma-Aldrich) for 2 minutes, blue-stained with 0.1% sodium bicarbonate for 1 minute, washed with water, and counterstained with Eosin Y solution (Sigma-Aldrich) for 1 minute. Subsequently, antigen retrieval was performed in a 97 ℃ PT Link Dako pretreatment module using Novocastar epitope retrieval fluid pH6.0 for 30 minutes. The slides were brought to room temperature and washed with PBS. With 3% H₂O₂After neutralization of endogenous peroxidase and blocking with a specific protein blocker (Novocastra), the samples were incubated overnight with primary antibody at 4 ℃. After incubation with HRP conjugated secondary antibody for 45 minutes, color development with Diaminobenzidine (DAB), hematoxylin counter staining, dehydration, supernatant, mounting. The antibodies used were as follows: CD4(Abcam, ab203034) and CD8(Abcam, ab 203035).

The micrographs in FIG. 5 show that treatment with the polysaccharide extract or anti-PD-1 antibody alone only slightly increased CD8⁺And CD4⁺Frequency of T effector cells in tumor infiltrating lymphocytes, and combination therapy resulted in CD8⁺And CD4⁺Recruitment of lymphocyte infiltrates and a significant increase in activation.

Thus, the above results indicate that the combination of polysaccharide extract and PD-1 inhibitor (especially anti-PD-1 antibody) produces additive or synergistic effects in inhibiting the proliferation or growth of tumors (especially lung cancer) in a subject and enhances the immune system of the subject by increasing the number of tumor-infiltrating T cells. The present invention provides an effective method for treating cancer (such as lung cancer, particularly non-small cell lung cancer) in a subject.

Claims

1. A method of treating a subject having cancer, the method comprising administering to the subject an effective amount of a polysaccharide extract and an effective amount of a PD-1 inhibitor, wherein the polysaccharide extract comprises ginseng polysaccharide.

2. The method of claim 1, wherein the cancer is lung cancer.

3. The method of claim 1, wherein the cancer is non-small cell lung cancer.

4. The method of claim 1, wherein the polysaccharide extract is administered to the subject before, after, or simultaneously with a PD-1 inhibitor.

5. The method of claim 4, wherein the polysaccharide extract is administered to the subject daily during the administration.

6. The method of claim 1, wherein the polysaccharide extract is administered orally to the subject.

7. The method of claim 1, wherein the polysaccharide extract comprises polysaccharides from dendrobe, ginseng, astragalus, and poria.

8. The method of claim 1, wherein the PD-1 inhibitor is an anti-PD-1 antibody.

9. The method of claim 1, wherein the PD-1 inhibitor is administered to the subject by injection.

10. A method of increasing the number of tumor infiltrating lymphocytes in cancer cells of a subject, comprising administering to the subject an effective amount of a polysaccharide extract and an effective amount of a PD-1 inhibitor, wherein the polysaccharide extract comprises ginseng polysaccharide.

11. The method of claim 10, wherein the cancer cell is from lung cancer.

12. The method of claim 10, wherein the cancer cell is from non-small cell lung cancer.

13. The method of claim 10, wherein the polysaccharide extract comprises polysaccharides from dendrobe, ginseng, astragalus, and poria.

14. The method of claim 10, wherein the PD-1 inhibitor is an anti-PD-1 antibody.

15. The method of claim 10, wherein said tumor infiltrating lymphocytes are selected from the group consisting of CD8⁺T lymphocytes, CD4⁺T lymphocytes or a combination thereof.

16. The method of claim 10, wherein said tumor-infiltrating lymphocytes comprise CD8⁺T lymphocytes and CD4⁺T lymphocytes.

17. A combination therapy comprising a polysaccharide extract and a PD-1 inhibitor, wherein the polysaccharide extract comprises ginseng polysaccharides.

18. The combination therapy of claim 17, wherein the PD-1 inhibitor is an anti-PD-1 antibody.

19. The combination therapy of claim 17, wherein the polysaccharide extract comprises polysaccharides from dendrobe, ginseng, astragalus, and poria.

20. The combination therapy of claim 17, wherein the molecular weight of the ginseng polysaccharide is 100,000 to 500,000.