AU2018101488A4

AU2018101488A4 - Method and kit for treating cancer through combination therapy

Info

Publication number: AU2018101488A4
Application number: AU2018101488A
Authority: AU
Inventors: Wen-Luan Wendy Hsiao; Zhi-hong JIANG; Lai-Han Elaine LEUNG; Liang Liu; Lian-Xiang Luo
Original assignee: Macau University of Science and Technology
Current assignee: Macau University of Science and Technology
Priority date: 2018-10-05
Filing date: 2018-10-05
Publication date: 2018-11-08
Anticipated expiration: 2026-10-05
Also published as: CN111000859A

Abstract

A method of treating a subject suffering from cancer comprising administering an effective amount of a polysaccharide extract and an effective amount of a PD-1 inhibitor to the subject, wherein the polysaccharide extract comprises ginseng polysaccharides. A method of increasing the amount of tumor-infiltrating leukocytes in cancer cells in a subject; and a kit comprising the polysaccharide extract and the PD-1 inhibitor. -. Votcle(Po$) 1500 .AnibPD.I(1*rqftc) Fig. 1 Vehicle(PBS) Anti-PD-i Anti-PD-1+GP Fig. 2

Description

TECHNICAL FIELD

The present invention relates to a method of treating cancer, in particular, but not exclusively, through a combination therapy. The invention also relates to a kit for said method.

BACKGROUND OF THE INVETNION

Immunotherapy enhances a patient’s immune system to fight cancer and has been a source of promising new cancer treatments. Among the many immunotherapeutic strategies, immune checkpoint blockade has shown remarkable benefit in the treatment of various cancers. Immune checkpoint blockade increases antitumor immunity by blocking intrinsic down-regulators of immunity, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death 1 (PD-1) or its ligand, programmed cell death ligand 1 (PD-L1)2. These immune checkpoint inhibitors exert their effect by disrupting the interaction between the inhibitory receptor PD-1 and its ligand PD-L1, leading to functional recovery of antitumour lymphocytes. Several immune checkpoint-directed antibodies induce durable clinical responses for patients with various cancers and are approved by the Food and Drug Administration (Garon EB, et al. The New England journal of medicine 2015, 372(21): 20182028). However, 85% of unselected patients will not respond to treatment, and nearly all of those who do initially respond will eventually develop resistant disease, immune checkpoint blockade can have immune-related side effects.

Currently, some combination approaches have been applied in cancer treatment. For example, for patients with disease resistant to PD-1 blockade, the use of combination therapies offers the potential to target additional pathways to improve the proportion of patients achieving a response. ALT-803, an IL-15 superagonist, in combination with nivolumab improves survival in patients with metastatic non-small cell lung cancer. Among patients with advanced melanoma, significantly longer overall survival occurred with combination therapy with nivolumab plus ipilimumab or with nivolumab alone than with ipilimumab alone. However, in view of frequent resistance mechanisms, there remains a need for therapeutic approaches for treatment of cancer, in particular for those with less adverse effects and suitable for inhibiting PD-1.

SUMMARY OF THE INVENTION

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

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

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

The inventors unexpectedly found that the combination of polysaccharide extract and PD-1 inhibitor of the present invention is effective in inhibiting the growth and development of a tumor including a cancer. The combination can further boost the immune system of a subject by increasing the macrophage infiltration in particular CD4+ and CD8+ lymphocytes into the tumor or cancerous tissues. Accordingly, the inventors have developed a new approach to treat cancer or alleviate symptoms associated with cancer by enhancing the immune system, or delaying the progression of the pathogenic condition. The invention is exceptionally suitable for treating lung cancer such as non-small cell lung cancer and may reduce adverse side effects brought by chemotherapeutic compounds.

Other features and aspects of the invention will become apparent by consideration of the following detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a plot showing the size of the tumors in the animal model after treatment with the combination of polysaccharide extract (denoted as GP) and anti-PD-1 antibody (denoted as anti-PD-1) in an embodiment of the present invention, the polysaccharide extract alone, the anti-PD-1 antibody alone, or phosphate-buffered saline (PBS) as control group, respectively (*P < 0.05 and **P < 0.01 vs. Anti-PD-1 antibody treated group; mean ± SD).

Fig. 2 is a photo showing the size of the tumors in the animal model after the treatments in

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Fig. 1.

Fig. 3 is a plot showing the weight of the tumors after treatments in Fig. 1, data are presented as mean ± SD, *P < 0.05 vs. Anti-PD-1 antibody treated group.

Fig. 4 is a plot showing the body weight of the animal model measured during the treatments in an embodiment of the present invention.

Fig. 5 shows microscopic images of excised tumor tissues obtained from the animal model after treatment with anti-PD-1 antibody, polysaccharide extract (GP), or the combination of anti-PD-1 antibody and polysaccharide extract, compared to the control group treated with vehicle PBS. These images show the CD4 and CD8 positivity in the tissues. Scare bar, 400x.

DETAILED DESCRIPTION OF THE EMBODIMENTS

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

As used herein, “comprising” means including the following elements but not excluding others. “Essentially consisting of’ means that the material consists of the respective element along with usually and unavoidable impurities such as side products and components usually resulting from the respective preparation or method for obtaining the material such as traces of further components or solvents. “Consisting of’ means that the material solely consists of, i.e. is formed by the respective element. As used herein, the forms “a,” “an,” and “the,” are intended to include the singular and plural forms unless the context clearly indicates otherwise.

The present invention in the first aspect provides a method for treating tumor in particular cancer in a subject through a combination therapy. The method comprises a step of administering an effective amount of a polysaccharide extract and an effective amount of a PD-1 inhibitor to the subject.

The term “polysaccharide extract” refers to a mixture obtained from a material such as an animal, a plant or a fungus and the content of polysaccharides in the mixture is at least 50% by weight based on the total weight of the mixture. The content of polysaccharides in the polysaccharide extract is at least 50%, 60%, 70%, 80%, or 90% by weight based on the total

2018101488 05 Oct 2018 weight of the polysaccharide extract. Preferably, the polysaccharide extract comprises neutral polysaccharides, acidic polysaccharides or a combination thereof; or the polysaccharide essentially consists of neutral polysaccharides, acidic polysaccharides, or a combination thereof.

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

The polysaccharide extract of the invention is preferably obtained by extracting at least one plant material such as, but not limiting to, a plant-based traditional Chinese medicine. The polysaccharide extract preferably comprises ginseng polysaccharides which are polysaccharides extracted from ginseng. Ginseng refers to the root part of any plants in the genus Panax such as Panax ginseng, Panax notoginseng and Panax quinquefolius, and any processed root part of said genus. In an embodiment, the polysaccharide extract comprises ginseng polysaccharides from Panax ginseng.

In another embodiment, the polysaccharide extract further comprises polysaccharides from at least one of Dendrobii Caulis, Ginseng Radix et Rhizoma, Scutellariae Radix, and Poria. In other words, the polysaccharide extract is prepared by extracting and isolating polysaccharide fractions from Panax ginseng, Dendrobii Caulis, Ginseng Radix et Rhizoma, Scutellariae Radix, and Poria. In particular, the polysaccharides have a molecular weight of from 100,000 to 500,000.

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

- providing the above herbal materials in a solvent such as water or alcohol;

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

- keeping the boiled mixture at room temperature overnight or subjecting the boiled mixture to centrifugation under 3000 g for 30 min;

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

- subjecting the supernatant to precipitation with alcohol in particular absolute alcohol; and

- loading the resultant mixture to column chromatography.

In a particular embodiment where the above steps are performed, 200 g to 1000 g of Panax ginseng, 200 g to 1000 g of Dendrobii Caulis, 200 g to 1000 g of Ginseng Radix et Rhizoma,

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200 g to 1000 g of Scutellariae Radix, and 200 g to 1000 g of Poria are immersed in a solvent, and heated in particular boiled once for about 1 to 5 hours, 2 to 4 hours, or 3 hours for extraction.

Preferably, DEAD-cellulose column is used for column chromatography. The isolated fraction includes neutral polysaccharide fraction and acidic polysaccharide fraction. More preferably, the fractions are later subject to size-exclusion column to obtain polysaccharides in a molecular weight range of from 50,000 to 1,000,000, from 80,000 to 750,000, or from 100,000 to 500,000, preferably from 100,000 to 500,000 MW.

The PD-1 inhibitor (Programmed cell death protein-1 inhibitor) of the invention refers to any substance that can target PD-1 present on the surface of cells. Without being limited by theory, the blockade of PD-1 can activate the immune system of a cell or a subject to fight against cancer. Preferably, the PD-1 inhibitor is anti-PD-1 antibody in particular anti-PD-1 monoclonal antibody. In an embodiment, the anti-PD-1 antibody is commercially available, for example but not exclusively, purchased from Biocell, clone: RMP1-14, Catalog#BE0146.

The inventors unexpected found that the polysaccharide extract of the invention works well with the PD-1 inhibitor to enhance the anti-cancer effect. The combination of the polysaccharide extract and the PD-1 inhibitor significantly inhibits the growth and development of in vivo cancer cell and boosts the immune system in mice through improving the recruitment and activation of tumor-infiltrating leukocytes.

The expression “effective amount” and “effective dose” generally denote an amount sufficient to produce therapeutically desirable results, wherein the exact nature of the result varies depending on the specific disorder which is treated. In this invention, the disorder is cancer and therefore the result is usually an inhibition or suppression of the proliferation of the cancer cells, a reduction of cancerous cells or the amelioration of symptoms related to the cancer cells. Particularly, the size of the tumor will be reduced after treatment.

The effective amount of the polysaccharide extract and PD-1 inhibitor of the present invention may depend on the species, body weight, age and individual conditions of the subject and can be determined by standard procedures such as with cell cultures or experimental animals. In an embodiment, the dose of polysaccharide extract of the present invention for treating the subject may, for example, be 100 mg/kg, 150 mg/kg, 200 mg/kg, or 250 mg/kg. In an alternative embodiment, the dose of polysaccharide extract of the present

2018101488 05 Oct 2018 invention for treating the subject may, for example, be from about 5 mg/kg to 25 mg/kg, from 8 mg/kg to 20 mg/kg, or from 10 mg/kg to 15 mg/kg where the subject is a human. Also, the dose of PD-1 inhibitor for treating the subject may be from 5 mg/kg to 20 mg/kg, from 10 mg/kg to 15 mg/kg, 10 mg/kg or 15 mg/kg. Alternatively, the dose of PD-1 inhibitor may be 0.3 mg/kg to 2 mg/kg, 0.5 mg/kg to 1 mg/kg, or 0.8 mg/kg to 1 mg/kg wherein the subject is a human.

The subject can be a human or animal, in particular the subject is a mammal, preferably a human.

Each of the polysaccharide extract and the PD-1 inhibitor may be provided in a pharmaceutical composition comprising a physiological tolerable excipient. The skilled person is able to select suitable excipients depending on the form of the pharmaceutical composition and is aware of methods for manufacturing pharmaceutical compositions as well as able to select a suitable method for preparing the pharmaceutical composition depending on the kind of excipients and the form of the pharmaceutical composition. Particularly, each of the polysaccharide extract and the PD-1 inhibitor according to the invention can be present in solid, semisolid or liquid form to be administered by an oral, rectal, topical, parenteral or transdermal or inhalative route to a subject, preferably a human. Preferably, the polysaccharide extract is formulated in liquid such as suspension or solution form for administering to the subject via oral route. Preferably, the PD-1 inhibitor is formulated in liquid form for administering to the subject via injection such as, but not limiting to, intravenous, intraperitoneal, intramuscular, subcutaneous, and intradermal route.

The polysaccharide extract may be administered to the subject before, after or simultaneously with the PD-1 inhibitor in a course of medication. In an embodiment, the polysaccharide extract is administered to the subject daily in a course of medication and via oral route, while the PD-1 inhibitor is continually administered to the subject every 1 day, 2 days, 3 days, 4 days, or 5 days via injection. Particularly, the PD-1 inhibitor is administered to subject every 3 days via injection. The inventors found that the continually administration of the PD-1 inhibitor together with the daily administration of polysaccharide extract improve the immune system of the subject. Without intending to be limited by theory, the polysaccharide extract and PD-1 inhibitor boost the infiltration of leukocytes into tumors and trigger apoptosis in the cancer cells. The combination therapy can help to reduce adverse side effects and reduce the development of drug resistance due to prolonged administration of PD-1 inhibitor.

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The term “cancer” refers to or describes a physiological condition in subjects in which a population of cells are characterized by unregulated cell growth. The term “tumor” simply refers to a mass being of benign (generally harmless) or malignant (cancerous) growth. In an embodiment, the cancer is preferably selected from lung cancer, breast cancer, colorectal cancer, or kidney cancer. Preferably, the cancer is lung cancer in particular non-small cell lung cancer.

The present invention also pertains to a method of increasing the amount of tumor-infiltrating leukocytes in cancer cells or a tumor in a subject. Said method comprises a step of administering an effective amount of a polysaccharide extract and an effective amount of a PD-1 inhibitor to the subject. The polysaccharide extract and the PD-1 inhibitor are as described above.

Said “tumor-infiltrating leukocytes” refers to any white blood cells, particularly lymphocytes, which are capable of recognizing and targeting transformed cells before the cells form a tumor, thereby eliminating said cells. The increase in the amount of tumor-infiltrating leukocytes in cancer cells or a tumor generally shows an enhancement of recruitment and activation of tumor-infiltrating leukocytes in the cancer cells or tumor and thus results in a reduction of cancer cells or tumor size.

In an embodiment, the tumor-infiltrating leukocytes are T lymphocytes preferably selected from CD8+ lymphocytes, CD4+ lymphocytes, or a combination thereof. Particularly, the tumor-infiltrating leukocytes include CD8+ lymphocytes and CD4+ lymphocytes.

In a preferred embodiment, the polysaccharide extract comprises ginseng polysaccharides and preferably comprises polysaccharides from Dendrobii Caulis, Ginseng Radix et Rhizoma, Scutellariae Radix and Poria. The PD-1 inhibitor is an anti-PD-1 antibody. Particularly, the polysaccharide extract is administered to the subject via oral route on a daily basis while the PD-1 inhibitor is administered to the subject continually via injection every 1 day, 2 days, 3 days, 4 days or 5 days, or preferably every 3 days.

The inventors proved that the combination therapy according to the present invention effectively enhances the amount of tumor-infiltrating leukocytes in a tumor in a subject.

Therefore, the combination therapy can be used to improve the immune system of a subject against tumor.

Furthermore, the present invention also pertains to a kit suitable for the above methods. The

2018101488 05 Oct 2018 kit comprises a polysaccharide extract and a PD-1 inhibitor as described above. The polysaccharide extract comprises ginseng polysaccharides which preferably have a molecular weight from 10,000 to 50,000.

Preferably, the polysaccharide extract comprises polysaccharides from Dendrobii Caulis, Ginseng Radix et Rhizoma, Scutellariae Radix and Poria.

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

Accordingly, the present invention provides effective approach for treatment of cancer and a kit for said treatment. The invention further pertains to use of the polysaccharide extract and PD-1 inhibitor as described above in the preparation of medication for treatment of cancer.

The invention is further described with examples below for better understanding of the cores aspect of the invention. The examples below are not intended for limiting the scope of protection, but are used for illustrative purpose.

EXAMPLE 1

Preparation of polysaccharide extract and PD-1 inhibitor Traditional Chinese medicines including Shihu (Dendrobii Caulis), Renshen (Ginseng Radix et Rhizoma), Huangqin (Scutellariae Radix) and Fuling (Poria) are grounded, immersed in deionized water, and extracted with boiling water. Particularly, 200 g of each of the Traditional Chinese medicines are refluxed with 8-10 times of water (1600 - 2000 mL) for 2 hours. Extraction is then repeated.. After extraction, the mixture is placed at room temperature overnight or the mixture is centrifuged under 3000 g for 30 min. Then the supernatant is collected, concentrated and precipitated with 100 % ethanol at a volume ratio of 1:4. The residue is further treated with Sevag reagent to remove proteins, and the supernatant fraction is loaded on DEAE-cellulose column. The neutral polysaccharide fraction is eluted with water solvent, while the acidic polysaccharide fraction is eluted with 0.5M sodium chloride. Next, the fractions are concentrated, dialyzed and lyophilized for storage and further use. Each polysaccharide fraction is separated on size-exclusion column to produce polysaccharide in a molecular weight range of 100,000 to 500,000 MW. The composition of the sugars of polysaccharide is then examined by means of acid hydrolysis and HPLC analysis. All the polysaccharide fractions are combined to form a polysaccharide extract for subsequent use.

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As to the PD-1 inhibitor, Biocell anti-PD1 monclonal antibody (clone: RMP1-14, Catalog#BE0146) is used.

EXAMPLE 2

Antitumor effect on growth of tumor in animal model Animal model in particular 3LL tumor model was established to evaluate the antitumor effect of the combination therapy of the present invention, i.e. the polysaccharide extract and PD-1 inhibitor. Animal studies were performed according to institutional guidelines. 3LL cells (2* 10⁶) were injected subcutaneously into C57BL/6 mice at age 8-10 weeks to establish the animal model, i.e. Lewis lung carcinoma (LLC) in C57BL/6 mice. On day 7, melanomabearing mice with similar tumor size were randomly divided into four groups (n= 4), these groups were independently treated with vehicle i.e. PBS, anti-PD-1 antibody (10mg/kg) (RMP1-14, Bio X Cell, 250pg per injection), the polysaccharide extract of Example 1 (200mg/kg), or a combination of anti-PD-1 antibody and the polysaccharide extract. In particular, the anti-PD-1 antibody was injected intraperitoneally every three days in particular on Day 3, 6, 9, 12 and 15 days the polysaccharide extract was administered to the mice by gavage every day. The tumor size and survival rate were measured as mentioned above. Mice with tumor size larger than 20 mm at the longest axis were euthanized for ethical consideration.

Tumor volume (mm³) = length (mm) χ width (mm)² χ π/6.

Referring to Figs. 1 to 3, the administration of the combination therapy effectively inhibited the growth of tumor in the tumor bearing mice. The combination of the polysaccharide extract (GP) and the anti-PD-1 antibody results in a significant reduction in tumor growth, as compared with the effect of other treatments. With reference to Fig. 4, there are no substantial changes in body weight or observed toxicities during the treatment.

EXAMPLE 3

Effect on CD4+ and CD8+ lymphocytes activity The inventors further evaluated whether the combination therapy of the present invention can enhance the recruitment and activation of CD8+ and CD4+ lymphocytes cell infiltrate. Mice of each treatment group and control group were sacrificed on Day 18 and the tumor tissues were collected and fixed in 10% formalin. After fixation, the tissues were embedded in paraffin to prepare slides with sections (5 pm) of the tissues thereon. The sections were dewaxed in xylene, and dehydrated using a series of alcohol gradations to water. Then, the slides were stained with Mayer’s hematoxylin (Sigma-Aldrich) for 2 min, blued in 0.1% sodium bicarbonate for 1 min, washed in water and counterstained with Eosin Y solution

2018101488 05 Oct 2018 (Sigma-Aldrich) for 1 min. Subsequently, antigen retrieval was performed using Novocastra Epitope Retrieval Solutions pH 6.0 in a PT Link Dako pre-treatment module at 97 °C for 30 min. The slides were then brought to room temperature and washed in PBS. After neutralization of the endogenous peroxidase with 3% H2O2 and blocking by a specific protein block (Novocastra), the samples were incubated overnight at 4 °C with the primary antibodies. After incubation for 45 min with HRP-conjugated secondary antibody, the slides were visualized with diaminobenzidine (DAB) and counterstained with haematoxylin, dehydrated, cleared and mounted. The following antibodies were used: CD4 (Abeam, ab203034), and CD8 (Abeam, ab203035).

The microscopic images in Fig. 5 show that treatment with either polysaccharide extract or anti-PD-1 antibodies alone modestly increased the frequency of effector CD8⁺ and CD4⁺ T cells amongst tumor-infiltrating leukocytes, while the combination treatment results in a significant increase in the recruitment and activation of CD8+ and CD4+ lymphocytes cell infiltrate.

Accordingly, all the above results demonstrate that the combination of polysaccharide extract and PD-1 inhibitor in particular anti-PD-1 antibody produces an additive or synergistic effect in inhibiting the proliferation or growth of the tumor in particular 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 approach for treating cancer such as lung cancer in particular non-small cell lung cancer in a subject.

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Claims

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

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 the PD-1 inhibitor.

5. The method of claim 4, wherein the polysaccharide extract is administered to the subject daily in a course of medication.

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

7. The method of claim 1, wherein the polysaccharide extract comprises polysaccharides from Dendrobii Caulis, Ginseng Radix et Rhizoma, Scutellariae Radix 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 via injection.

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

11. The method of claim 10, wherein the cancer cells are from lung cancer.

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

13. The method of claim 10, wherein the polysaccharide extract comprises

2018101488 05 Oct 2018 polysaccharides from Dendrobii Caulis, Ginseng Radix et Rhizoma, Scutellariae Radix and Pori a.

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

15. The method of claim 10, wherein the tumor-infiltrating leukocytes are T lymphocytes selected from CD8+ lymphocytes, CD4+ lymphocytes, ora combination thereof.

16. The method of claim 10, wherein the tumor-infiltrating leukocytes include CD8+ lymphocytes and CD4+ lymphocytes.

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

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

19. The kit of claim 10, wherein the polysaccharide extract comprises polysaccharides from Dendrobii Caulis, Ginseng Radix et Rhizoma, Scutellariae Radix and Poria.

20. The kit of claim 10, wherein the ginseng polysaccharides have a molecular weight from 100,000 to 500,000.

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Drug trcatmeat(Days)

Fig. 1

Vehicle(PBS) Anti-PD-1 r * ► W * GP IK Anti-PD-l+GP '* t £ < · eL_1 fc 7 x » |i

Fig. 2

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Fig. 3

Fig. 4

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