CN113134080A

CN113134080A - Application of anti-PD-1 antibody and furoquintinib combination in preparation of medicine for treating cancer

Info

Publication number: CN113134080A
Application number: CN202010052280.6A
Authority: CN
Inventors: 闫洪滨; 王斯斯; 谢帆; 戴秋曦; 陈文会; 王倩; 郭倩
Original assignee: GENOR BIOPHARMA CO Ltd; Hutchison Medipharma Ltd
Current assignee: GENOR BIOPHARMA CO Ltd; Hutchmed Ltd
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2021-07-20
Also published as: CN115003332A; WO2021143799A1

Abstract

The invention discloses an application of an anti-PD-1 antibody or an antigen binding fragment thereof and furoquintinib or a pharmaceutically acceptable salt thereof in preparation of a medicament for treating cancer. The anti-PD-1 antibody and the furoquintinib have a certain synergistic effect when being used together, compared with the corresponding single drug, the anti-PD-1 antibody has an obvious effect of inhibiting the growth of tumors, and during the drug administration, the experimental animals have good tolerance and no adverse reaction.

Description

Application of anti-PD-1 antibody and furoquintinib combination in preparation of medicine for treating cancer

Technical Field

The invention relates to the field of medicines, and in particular relates to an application of an anti-PD-1 antibody or an antigen binding fragment thereof and furoquintinib or a pharmaceutically acceptable salt thereof in preparation of a medicine for treating cancer.

Background

Vascular endothelial cell growth factor (VEGF) is one of the major inducing factors currently found to be associated with tumor angiogenesis, and binding to VEGF receptor (VEGFR) can activate angiogenesis, leading to the continuous growth and metastatic spread of tumors. Blocking VEGF/VEGFR signal transduction pathway can inhibit angiogenesis, thereby inhibiting tumor growth and achieving the effect of anti-tumor. At present, a plurality of VEGF/VEGFR inhibitors are available, including monoclonal antibody drugs bevacizumab and small molecule kinase inhibitor drugs sunitinib, sorafenib, ranvatinib, regorafenib, axitinib, furacitinib and the like.

VEGFR small molecule inhibitor furquintinib (Fruquintinib) disclosed in patent number CN101575333B, chemical name of which is 6- (6, 7-dimethoxyquinazoline-4-oxo) -N, 2-dimethylbenzofuran-3-formamide, molecular formula of which is C₂₁H₁₉N₃O₅The structural formula is shown as the following formula:

furquintinib is a highly selective tumor angiogenesis inhibitor, the main action targets of the furquintinib are VEGFR kinase families VEGFR1, VEGFR2 and VEGFR3, and the furquintinib can inhibit VEGFR phosphorylation, so that tumor angiogenesis is inhibited, and tumor growth is finally inhibited.

Programmed Death receptor (PD-1) and ligand PD-L1/L2 are important T-cell negative regulation immune check sites, regulate the peripheral immune tolerance of the organism and play an important role in peripheral immune balance. The cancer cells can be combined with PD-1 receptors on the surfaces of T-cells by high-expression PD-L1 to inhibit the activation of immune cells, and induce tumor immune tolerance PD-1/PD-L1 to play an important role in the processes of tumor immune escape and tumor microenvironment formation. The monoclonal antibody drug represented by the targeted PD-1 and the ligand PD-L1 thereof blocks the negative immune check site PD1/PD-L1 to activate and proliferate T-cells, can reverse the tumor immunosuppressive microenvironment, enhance the anti-tumor immune response, and realize tumor immunotherapy, thereby effectively inhibiting the growth of tumors. The anti-PD-1 antibody (CN106573052A discloses the antibody and its preparation method, hereinafter also referred to as GB226) of products of Jia and biological medicine industry is in clinical stage II at present, has good safety and has effective anti-tumor effect.

In the process of treating cancer, the single administration treatment effect has certain limitation, the drug effect is not obvious, and the drug resistance is easy to generate. For example, metastatic colorectal cancer patients and EGFR-TKI drug-resistant non-small cell lung cancer after standard treatment failure have limited treatment means at present, and new treatment methods and drugs are urgently needed to improve the curative effect and prolong the life of the patients.

Clinical studies on the combination of anti-PD-1 antibodies and VEGFR inhibitors for the treatment of cancer are currently under development, but no information is approved for marketing. WO2015119930 discloses the use of an anti-PD-1 antibody in combination with axitinib, WO2015088847 discloses the use of an anti-PD-1 antibody in combination with pazopanib, and CN105960415A discloses the use of an anti-PD-1 antibody in combination with axitinib.

Disclosure of Invention

The invention provides an anti-PD-1 antibody or an antigen-binding fragment thereof and furoquintinib or a pharmaceutically acceptable salt thereof, which are combined to be used for preparing a medicament for treating cancer.

Preferably, the above-mentioned anti-PD-1 antibody or an antigen-binding fragment thereof comprises a light chain CDR1(LCDR1) sequence which is the amino acid sequence shown in SEQ ID NO. 1 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO. 1, a light chain CDR2(LCDR2) sequence which is the amino acid sequence shown in SEQ ID NO. 2 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO. 2, a light chain CDR3(LCDR3) sequence which is the amino acid sequence shown in SEQ ID NO. 3 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO. 2, a heavy chain CDR1(HCDR1) sequence which is the amino acid sequence shown in SEQ ID NO. 4 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO. 4, a heavy chain CDR2(HCDR2) sequence which is the amino acid sequence shown in SEQ ID NO. 5 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO. 5, and a heavy chain CDR3(HCDR3) sequence which is the amino acid sequence shown in SEQ ID NO. 6 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO. 6.

Preferably, the anti-PD-1 antibody or an antigen-binding fragment thereof is a humanized antibody or an antigen-binding fragment thereof.

Preferably, the humanized antibody or antigen binding fragment thereof comprises a light chain variable region sequence that is the amino acid sequence set forth in SEQ ID NO. 7 or that is at least 80% homologous to the amino acid sequence set forth in SEQ ID NO. 7.

Preferably, the above-mentioned humanized antibody or antigen-binding fragment thereof comprises a heavy chain variable region sequence which is the amino acid sequence shown in SEQ ID NO. 8 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO. 8.

Preferably, the heavy chain sequence of the humanized antibody is shown as SEQ ID NO. 9, and the light chain sequence is shown as SEQ ID NO. 10.

Preferably, the cancer expresses PD-L1.

Preferably, the cancer is lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, intestinal cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, stomach cancer, testicular cancer, salivary gland cancer, thyroid cancer, thymus cancer, epithelial cancer, head and neck cancer, stomach cancer, pancreatic cancer, prostate cancer, renal cancer or a combination thereof.

Preferably, the cancer is colon cancer, rectal cancer, carcinoma of large intestine, carcinoma of small intestine, and non-small cell lung cancer.

More preferably, the cancer is recurrent or metastatic non-small cell lung cancer, metastatic colon cancer.

Preferably, the anti-PD-1 antibody is used in an amount of 0.1 to 40mg/kg or a fixed dose of 100 to 400 mg.

More preferably, the anti-PD-1 antibody is used in an amount of 3mg/kg or a fixed dose of 210 mg.

Preferably, the anti-PD-1 antibody is administered once every two weeks.

Preferably, the anti-PD-1 antibody is used in an amount of 5 mg/kg. Preferably, the administration is twice weekly.

More preferably, the anti-PD-1 antibody is GB226 administered in an amount of 3mg/kg per patient's body weight or a fixed dose of 210mg administered once every two weeks.

Preferably, the amount of furoquintinib is 0.1-10mg/kg or a fixed dose of 0.1-20mg is used.

More preferably, furoquintinib is used in an amount of 1mg/kg, 2mg/kg, or alternatively a fixed dose of 2mg, 3mg, 4mg or 5mg is used.

More preferably, furoquintinib is used in a fixed dose of 2mg, 3mg, 4mg or 5 mg.

More preferably, furoquintinib is administered once daily. More preferably, the administration is for three weeks, the rest for one week, or the administration is for two weeks, the rest for one week.

More preferably, furoquintinib is administered in a fixed dose of 2mg once daily for three weeks, and off for one week.

More preferably, furoquintinib is administered in a fixed dose of 3mg once daily for three weeks, and off for one week.

More preferably, furoquintinib is administered in a fixed dose of 4mg once daily for three weeks, and off for one week.

More preferably, furoquintinib is administered in a fixed dose of 5mg once daily for three weeks, and off for one week.

Preferably, the anti-PD-1 antibody or antigen-binding fragment thereof and furoquintinib or a pharmaceutically acceptable salt thereof are administered during the same dosing cycle.

The invention also provides a kit, which contains the anti-PD-1 antibody or the antigen binding fragment thereof and furacitinib or pharmaceutically acceptable salts thereof.

The invention also provides an anti-PD-1 antibody or an antigen binding fragment thereof and furoquintinib or pharmaceutically acceptable salts thereof which are used for preparing medicaments for treating tumors.

The invention also provides the anti-PD-1 antibody or the antigen binding fragment thereof and the furoquintinib or the pharmaceutically acceptable salt thereof which are used as medicines for reducing adverse drug reactions, wherein the adverse drug reactions are selected from the group consisting of the anti-PD-1 antibody and the furoquintinib or the pharmaceutically acceptable salt thereof.

The invention also provides the anti-PD-1 antibody or the antigen-binding fragment thereof and furquintinib or the pharmaceutically acceptable salt thereof which are combined to be used as medicines for reducing the single administration dosage of the anti-PD-1 antibody and/or the single administration dosage of the furquintinib or the pharmaceutically acceptable salt thereof.

Also provided in the present invention is a method of reducing the dose of an anti-PD-1 antibody or antigen-binding fragment thereof administered alone and/or the dose of furoquintinib or a pharmaceutically acceptable salt thereof administered alone, comprising administering to a patient the above-described anti-PD-1 antibody in combination with furoquintinib or a pharmaceutically acceptable salt thereof.

The present invention also provides a method of treating a tumor/cancer comprising administering to a patient the above-described anti-PD-1 antibody or antigen-binding fragment thereof and furoquintinib or a pharmaceutically acceptable salt thereof.

Drawings

FIG. 1 shows a growth curve of a mouse colorectal cancer MC38 model after the start of treatment, which illustrates the effect of the anti-PD-1 antibody of the present invention administered alone or in combination with furoquintinib on the tumor volume of the mouse colorectal cancer MC38 model, and it can be seen from the graph that the inhibition rates of tumor growth in the GB226 group, the Fruquintinib high dose group, the Fruquintinib low dose group, the GB226+ Fruquintinib high dose group, and the GB226+ Fruquintinib low dose group are 90%, 73%, 56%, 94%, 93%, respectively, at the end of the control group (PG-D17), and the tumor volume of each treatment group is significantly lower than that of the control group (p < 0.05). Before the experiment (PG-D24), the tumor volume of the GB226+ Fruquintinib high-dose group and the GB226+ Fruquintinib low-dose group is obviously lower than that of the corresponding GB226 and Fruquintinib single-drug groups (p is less than 0.05), and the combined drug groups have no obvious difference (p is 0.885).

Fig. 2 shows the body weight change curve of the experimental animals after the start of treatment, which illustrates the body weight change curve of the experimental animals after the administration of the anti-PD-1 antibody of the present invention and furacitinib alone or in combination, and it can be seen from the figure that during the treatment period, the tumor-bearing mice show good tolerance to the test substances furacitinib, GB226 and the combination thereof, and the body weights of the mice in each group are normal, no abnormal behavior is shown, and the general state is good.

FIG. 3 shows the percent change in body weight of the experimental animals after initiation of treatment, which illustrates the percent change in body weight of the experimental animals after administration of an anti-PD-1 antibody of the invention, either alone or in combination with furoquintinib. It can be seen from the figure that during the treatment period, the tumor-bearing mice show good tolerance to the test substances of furoquintinib, GB226 and the combination thereof, and the weight of each group of mice is normal, no abnormal expression exists, and the general state is good.

Detailed Description

In the description of the present invention, the sequences of LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 contained in the above-described anti-PD-1 antibody are shown in table 1 below.

TABLE 1

Name (R)	Numbering	Sequence of
			LCDR1	SEQ ID NO:1	RASESVDNYGYSFMN
LCDR2	SEQ ID NO:2	RASNLES
			LCDR3	SEQ ID NO:3	QQSNADPT
HCDR1	SEQ ID NO:4	NFGMN
			HCDR2	SEQ ID NO:5	WISGYTREPTYAADFKG
HCDR3	SEQ ID NO:6	DVFDY

In the present invention, the sequences of the light chain and heavy chain variable regions of the above humanized antibody are shown in Table 2 below.

TABLE 2

In describing the present invention, the full-length heavy chain and full-length light chain sequences of the above humanized antibodies are shown in table 3 below.

TABLE 3

In the use described in the present invention, the cancer is a cancer expressing PD-L1. Such cancers include, but are not limited to, lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, intestinal cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, stomach cancer, testicular cancer, salivary gland cancer, thyroid cancer, thymus cancer, epithelial cancer, head and neck cancer, stomach cancer, pancreatic cancer, prostate cancer, renal cancer, or combinations thereof. In a preferred embodiment of the present invention, the cancer is colorectal cancer. In a preferred embodiment of the present invention, the cancer is metastatic colon cancer. In a preferred embodiment of the present invention, the cancer is non-small cell lung cancer (NSCLC). In a more preferred embodiment of the present invention, the non-small cell lung cancer is recurrent or metastatic non-small cell lung cancer (NSCLC) with EGFR-sensitive mutation that fails in Epidermal Growth Factor Receptor (EGFR) -Tyrosine Kinase Inhibitor (TKI) treatment. In a preferred embodiment of the present invention, the cancer is small intestine cancer. In a preferred embodiment of the present invention, the cancer is colon cancer.

The term "pharmaceutically acceptable salt" as used herein refers to a salt of furoquintinib that is non-toxic, biologically tolerable or otherwise biologically suitable for administration for the treatment or prevention of disease. Including but not limited to acid addition salts or base addition salts, such as: acid addition salts of furoquintinib with inorganic acids, such as hydrochloride, hydrobromide, carbonate, bicarbonate, phosphate, sulfate, sulfite, nitrate, and the like; and acid addition salts of furoquintinib with organic acids, for example formates, acetates, malates, maleates, fumarates, tartrates, succinates, citrates, lactates, methanesulfonates, p-toluenesulfonates, 2-hydroxyethanesulfonates, benzoates, salicylates, stearates and with compounds of formula HOOC- (CH)₂)_nSalts with alkanedicarboxylic acids of-COOH (wherein n is 0 to 4), and the like.

In the uses described herein, the anti-PD-1 antibody may be administered in an amount of 0.1 to 40mg/kg, preferably 0.5 to 20mg/kg, more preferably 1 to 10mg/kg, most preferably 3mg/kg or 5 mg/kg; for adult humans, fixed doses may also be used, for example 100-400mg each time, preferably 150-300mg, most preferably 210 mg. The frequency of administration of the anti-PD-1 antibody may be once every 1 to 3 weeks, preferably once every 2 weeks.

In the application described in the invention, the administration dose of the furoquintinib can be 0.1-10mg/kg, and preferably 0.5-5 mg/kg. For adult humans, a fixed dose may be used, for example 0.1-20mg, preferably 1-10mg, more preferably 2mg, 3mg, 4mg or 5 mg. The dosing frequency of furoquintinib may be once daily, more preferably three weeks, and one week off.

In the use described herein, the anti-PD-1 antibody is preferably administered by injection, for example, subcutaneously or intravenously, and the anti-PD-1 antibody is formulated in an injectable form prior to injection. The injectable form of the anti-PD-1 antibody may be an injection solution or a lyophilized powder injection, which comprises the anti-PD-1 antibody, a buffer, a stabilizer, and optionally further comprising a surfactant. The buffer can be one or more selected from acetate, citrate, succinate and phosphate. The stabilizer may be selected from sugars or amino acids, preferably disaccharides, such as sucrose, lactose, trehalose, maltose. The surfactant is selected from polyoxyethylene hydrogenated castor oil, glycerin fatty acid ester, and polyoxyethylene sorbitan fatty acid ester, preferably the polyoxyethylene sorbitan fatty acid ester is polysorbate 20, 40, 60, or 80, and most preferably polysorbate 20. Most preferred injectable forms of anti-PD-1 antibodies comprise an anti-PD-1 antibody, acetate buffer, trehalose, and polysorbate 20. Furoquintinib is preferably administered orally, e.g. it can be formulated for administration as a tablet or capsule.

In the use described herein, "combination" is a mode of administration that includes administration of the two drugs sequentially, or simultaneously, in each case, and by "simultaneously" herein is meant administration of the anti-PD-1 antibody and furquintinib in the same administration cycle, e.g., within 2 days, or within 1 day. By "sequential" administration, it is meant to include administration of the anti-PD-1 antibody and furoquintinib separately over different administration cycles. These administration modes are all the combinations of the above administration modes.

In the application described by the invention, when the anti-PD-1 antibody is used in combination with furquintinib, adverse drug reactions caused by the anti-PD-1 antibody and/or the furquintinib can be reduced. The above adverse reactions can be selected from vascular adverse reactions, gland hypofunction, skin adverse reactions, respiratory system adverse reactions, liver adverse reactions, endocrine adverse reactions, digestive system adverse reactions, kidney adverse reactions, fatigue, and fever; said angio-related adverse reactions are selected from hemangioma, vasculitis, lymphangioma, and said glandular hypofunction is selected from hypothyroidism, hypoparathyroidism, pancreatopathy, and prostate hypofunction; said adverse skin reactions are selected from the group consisting of pruritus, urticaria, rashes, and toxic epidermal necroses; the above-mentioned adverse reaction of respiratory system is selected from pneumonia, bronchitis, chronic obstructive pulmonary disease, pulmonary fibrosis; the adverse reactions related to the liver can be selected from hepatitis and abnormal liver function; the above endocrine-related adverse reactions can be selected from type I diabetes, type II diabetes, and hypoglycemia; the kidney-related adverse reactions can be selected from nephritis and renal failure; the above digestive system adverse reaction can be selected from diarrhea, nausea, emesis, enteritis, and constipation. More preferably, the adverse drug reactions are selected from the group consisting of hemangioma, hypothyroidism, and parathyroid function.

In the method for reducing the dose of the anti-PD-1 antibody and/or the dose of furoquintinib administered alone, which is used in combination with the anti-PD-1 antibody, the administration dose of furoquintinib is 10% -100%, preferably 10% -75%, more preferably 75%, 50%, 25%, 12.5% of the dose of furoquintinib administered alone. When used in combination with furoquintinib, the anti-PD-1 antibody is administered in an amount of 10% to 100%, preferably 10% to 50%, of the amount of the anti-PD-1 antibody administered alone.

In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the invention is further explained by combining the embodiments.

Example 1

In vivo pharmacodynamic studies of furoquintinib (Fruquintinib) in combination with anti-PD-1 antibodies on a PD-1 humanized MC38 mouse tumor model.

1. Raw material information and source

1.1 raw materials information

1.1.1 Fuoquintinib (Fruquintinib): the chemical name is 6- (6, 7-dimethoxy quinazoline-4-oxo) -N, 2-dimethyl benzofuran-3-formamide with the molecular formula of C₂₁H₁₉N₃O₅Its preparation is disclosed in the patent CN 101575333B.

The structural formula is shown as the following formula:

1.1.2 GB226 sequence information:

1.2 sources of raw materials

1.2.1 Fuoquintinib (Fruquintinib):

providing a unit: jiekang (Beijing) pharmaceutical science and technology Co., Ltd

Supplier: hequan pharmaceutical Co Ltd

Batch: C13052210-AF18003M

Shape: white powder

Quantity: 50 mg/bag

1.2.2 anti-PD-1 antibody: the anti-PD-1 antibody is prepared according to the method disclosed by CN106573052A, and the heavy chain and light chain sequences of the anti-PD-1 antibody are shown as SEQ ID NO. 9 and SEQ ID NO. 10 of the invention.

Providing a unit: jiahe biological pharmaceutical Co Ltd

Batch: RD-201805001

The characteristics are as follows: tongming liquid

Quantity: 70 mg/bottle, 3 bottles

2. Raw material preparation method and storage conditions

anti-PD-1 antibody (10 mg/ml): accurately measuring 0.3ml of sample stock solution in a glass bottle by using a microsyringe, and fully and uniformly mixing another 5.7ml of PBS in the glass bottle until the final concentration is 0.5mg/ml, and preparing the sample in situ when the sample is used;

furoquintinib (Fruquintinib): accurately weighing 5.6mg of the furoquintinib sample in a glass bottle, weighing 28ml of 0.5% CMC-Na in the glass bottle, carrying out vortex oscillation for 1-3 minutes, carrying out ultrasound for 15 minutes by using an ultrasonic cleaner, fully and uniformly mixing until the final concentration is 0.2mg/ml, preparing once per week, subpackaging, and storing at 4 ℃;

3. laboratory animals and rearing management

3.1. Laboratory animal

Mus Musculus, huPD-1C57BL/6 (mouse, PD-1), 6-8 weeks old, were female, weighing 18-23g, for a total of 62 (48 subjects in the group); the experimental animal is provided by Beijing Weitonglihua experimental animal technology Limited, and the production license number is as follows: SCXK (Jing) 2016-: 1100111911048361.

3.2 Breeding management

Experimental animals were housed in a SPF-grade constant temperature and humidity laminar flow clean room with independent ventilation cages IVC, one cage per 4 rats, with padding changed twice per week, each cage having a cage label indicating the number of animals, sex, strain, receiving time, group and start time of the experiment. The temperature and humidity are controlled within the range of (23 +/-3) DEG C/40-70%. SPF mouse material, cobalt 60 radiation disinfection, drinking water is ultra-filtration purified water, and animals can freely take sterile food and drinking water after autoclaving treatment. Animals were numbered with ear perforations.

4. Experimental methods

4.1 cell culture

MC38 mouse colon cancer cells (YK-CL-256-02) were purchased from Purpurin Biotech (Beijing) Inc. (Biovector NTCC Inc.), using DEME medium containing inactivated 10% fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin and 2mM glutamine at 37 ℃ in 5% CO₂The culture box is used for culturing the tumor cells, bottle-dividing passage is carried out after the cells grow full every 3 to 4 days, and the tumor cells in logarithmic growth phase are used for inoculation of in vivo tumors.

4.2 tumor cell inoculation and grouping

PBS resuspended MC38 tumor cells at a concentration of 1X 10⁷Perml, 100. mu.l/mouse inoculated subcutaneously into the right anterior flank of PD-1 humanized C57BL/6 mouse, and the tumor growth reached 88m³The left and the right are administered in groups (marked as PG-D0 on the same day),for a total of 6 groups of 8, the specific dosing schedule is shown in the following table:

TABLE 4 dosing regimen

Note: the administration volume is 10 mul/g according to the animal body weight, and the administration amount can be adjusted when the body weight is reduced by 15-20%;

i.p.: performing intraperitoneal injection; p.o.: intragastric administration; biw × 4: 2 times per week for a total of 4 times; qd × 18/23/26: the administration is performed 1 time per day for 18, 23 and 26 days (times).

4.3 measurement and Experimental indices of mouse body weight

The tumor volume was measured 3 times per week using a vernier caliper and the mouse body weight was weighed with an electronic balance to measure the long and short diameters of the tumor, the volume calculation formula was: volume is 0.5 x long diameter x short diameter². The results of the measurements are shown in Table 5. The T/C value was calculated from the Tumor Volume, where T is the mean Relative Tumor Volume (RTV) of each subject treatment group, and C is the mean Relative Tumor Volume (RTV) of the control group. The T/C value is the ratio of the tumor volume after administration to that before administration. The tumor growth inhibition ratio (%) (1-T/C) × 100%. In the experimental process, in order to meet the ethical specifications of experimental animals, for example, the tumor volume is more than 2000mm³The mice were euthanized. Therefore, the control group sacrificed mice at PG-D17; the furoquintinib low dose group mice were sacrificed at PG-D22; the GB226 group, the furoquintinib high dose group, the GB226+ furoquintinib high dose group, and the GB226+ furoquintinib low dose group all sacrificed mice at PG-D25. After euthanasia, the mice were weighed for tumors. Since tumor weight was only weighed after mice were sacrificed, tumor weight was measured only once for each dosing group and control group. The tumor weight inhibition rate is calculated by the formula of 1-the ratio of the tumor weight of the administration group to the tumor weight of the control group, wherein the tumor weight of the control group is PG-D17The tumor weight of the furquintinib low-dose group is based on PG-D22, and the tumor weight of the other administration groups is based on PG-D25. The results of the measurements are shown in Table 6.

5. Results of the experiment

5.1 tumor growth inhibition results

According to the tumor volume (see table 5), the growth curve of the mouse colorectal cancer MC38 model after the start of treatment (see fig. 1) and the tumor weight (see table 6), the results show that at the end of the control group (PG-D17), the tumor growth inhibition rates of the GB226 group, the Fruquintinib high-dose group, the Fruquintinib low-dose group, the GB226+ Fruquintinib high-dose group and the GB226+ Fruquintinib low-dose group are respectively 90%, 73%, 56%, 94% and 93%, and the tumor volume of each treatment group is significantly lower than that of the control group (p < 0.05). Before the experiment (PG-D24), the tumor volume of the GB226+ Fruquintinib high-dose group and the GB226+ Fruquintinib low-dose group is obviously lower than that of the corresponding GB226 and Fruquintinib single-drug groups (p is less than 0.05), and the combined drug groups have no obvious difference (p is 0.885).

Tumor weight change (see table 6): the tumor weight inhibition rates of the mice in the GB226 group, the Fruquintinib high-dose group, the Fruquintinib low-dose group, the GB226+ Fruquintinib high-dose group and the GB226+ Fruquintinib low-dose group are respectively 40.1%, 19.35%, 10.57%, 83.33% and 83.30%. The inhibition rate of the tumor weight can find that the combination has obvious synergistic effect.

TABLE 6 tumor suppression (tumor weight) of test substances on mouse colorectal cancer MC38 model

Note:^a.mean ± standard error.

Mice in the Vehicle control group and the Fruquintinib low dose (1mg/kg) group ended early in PG-D17 and PG-D22, respectively, due to tumor overgrowth.

5.2 mouse safety Effect

The experimental animals had weight changes after the start of treatment (see fig. 2, fig. 3). The results show that during the treatment period, the tumor-bearing mice show good tolerance to the test substances of furacitinib, GB226 and the combination thereof, and the weight of each group of mice is normal, the abnormal expression is avoided, and the general state is good.

6. Summary of the invention

The test drugs GB226(5mg/kg), the high dose of furoquintinib (2mg/kg), the high dose of GB226+ furoquintinib (2mg/kg) and the low dose of GB226+ furoquintinib (1mg/kg) have obvious anti-tumor effect on a PD-1 humanized MC38 mouse tumor model, effectively inhibit tumor growth, and the anti-tumor effect of the combined drug of furoquintinib and GB226 is obviously superior to that of corresponding single-drug therapy. This shows that the anti-PD-1 antibody GB226 and furoquintinib have a certain synergistic effect in combination, and compared with the corresponding single drug, the effect of inhibiting tumor growth is obvious. The animals have good tolerance and no adverse reaction during the administration of GB226 and furacitinib.

Example 2

Phase Ib clinical test of anti-PD-1 antibody and furoquintinib for treating metastatic colon cancer

Purpose of study

The main purpose is：

Evaluation of the safety and tolerability of GB226 in combination with furoquintinib for the treatment of metastatic colorectal cancer (mCRC)

Obtaining the recommended dose for future clinical studies of GB226 in combination with furoquintinib for the treatment of metastatic colorectal cancer (mCRC)

For a second purpose：

Evaluation of Pharmacokinetic (PK) profiles of GB226 in combination with furoquintinib

Preliminary evaluation of the antitumor Activity of GB226 in combination with Fuquintinib for the treatment of mRC

Evaluation of the immunogenicity of GB226

Exploratory object：

Researches on the correlation of the expression of programmed cell death protein ligand-1 (PD-L1) in tumor tissues of mCRC patients, and the clinical curative effects of biomarkers such as microsatellite instability (MSI) and/or mismatch repair deficiency (dMMR) and tumor mutation load (TMB) and GB226 combined furquintinib

And (3) inclusion standard:

at least 21 patients with metastatic colorectal cancer who failed treatment at one or more lines were planned to be enrolled.

The following criteria are to be met:

1. age 18 to 75 years, with no restriction;

2. understanding the experimental procedures and content and voluntarily signing written informed consent;

3. patients with histo/pathologically confirmed colorectal cancer;

4. metastatic colorectal cancer patients who have failed first line or more therapy. Treatment failure refers to disease progression or intolerance of toxicity after receiving treatment for more than or equal to 1 cycle; or relapse during adjuvant chemotherapy/neoadjuvant therapy or within 6 months after the treatment is finished;

an ECOG score of 0-1;

6. the expected survival is more than or equal to 3 months;

7. at least one measurable and evaluable tumor lesion (according to RECIST 1.1 criteria) is present;

8. before the first study, systemic chemotherapy, targeted therapy or other anti-tumor biotherapeutics (tumor vaccines, cytokines or growth factors for tumor control) are completed for at least 4 weeks (oral fluorouracil is stopped for at least 2 weeks); systemic or local palliative radiotherapy is completed for at least 4 weeks; has not received anti-angiogenesis small molecule targeted drugs;

9. prior to first study administration, systemically administered corticosteroid drug (prednisone >10 mg/day or equivalent) has been discontinued for at least 2 weeks;

10. prior to the first study, major surgery requiring general anesthesia must have been completed for at least 8 weeks; procedures requiring local/epidural anesthesia must have been completed for at least 4 weeks;

11. the routine requirement of blood is that hemoglobin is more than or equal to 90g/L (no transfusion is allowed within 14 days before the routine examination of baseline blood), and the function of neutrophil is more than or equal to 1.510⁹L (no recombinant human granulocyte-colony stimulating factor support treatment within 14 days before the baseline blood routine examination) and 100X 10 or more platelets⁹L (no supportive treatment with recombinant human thrombopoietin or blood transfusions, etc. within 14 days prior to baseline blood routine);

12. serum creatinine is less than or equal to 1.5 × ULN or calculated creatinine clearance is more than or equal to 60mL/min (Cockcroft-Gault formula), and urine protein is less than 2+ or less than 1.0 g/L. For patients with urine protein more than or equal to 2+ or more than or equal to 1.0g/L at baseline, urine protein quantitative detection for 24 hours must be less than or equal to 1.0g/L for selection;

13. total bilirubin ≦ 1.5 × ULN (unless Gilbert's syndrome is demonstrated), aspartate Aminotransferase (AST) and alanine aminotransferase (ALT ≦ 2.5 × ULN (liver transfer patients allow AST and/or ALT ≦ 5 × ULN);

14. thyroid function index: thyroid Stimulating Hormone (TSH), free thyroxine (FT3/FT4) are in the normal range; if the TSH is not in the normal range, FT3/FT4 is in the normal range and can be grouped;

15. adverse reactions caused by previous treatments are restored to grade 1 and below before entering the group (except for alopecia and neurotoxicity not more than grade 2 caused by chemotherapeutic drugs);

16. the non-pregnant women are confirmed within 7 days before administration, and the male or female in the birth period needs to agree to take medically approved effective contraceptive measures in the whole test period and within 6 months after the test is finished;

17. patients can follow up on schedule, can communicate well with researchers, and can complete studies in accordance with the study prescription.

Exclusion criteria:

if any of the following criteria are met, the study must be excluded.

1. Active Central Nervous System (CNS) metastases, including symptomatic brain metastases or meningeal metastases or spinal cord compression, etc.; asymptomatic brain metastases can be enrolled (no progression after at least 4 weeks of radiotherapy and/or no neurological symptoms or signs after surgical resection, without treatment with glucocorticoids, antiepileptics, anticonvulsants or mannitol);

2. a patient who has previously suffered from other malignancies (with the exception of cured cervical carcinoma in situ and basal cell carcinoma of the skin) must not participate in the study unless he/she has complete remission for at least 2 years prior to enrollment and does not need to receive other treatments or other treatments during the study;

3. there is an active, known history of autoimmune diseases, including but not limited to systemic lupus erythematosus, psoriasis, rheumatoid arthritis, inflammatory bowel disease, hashimoto's thyroiditis, and the like, with the exception of: type I diabetes, hypothyroidism which can be controlled by hormone replacement therapy alone, skin disorders which do not require systemic treatment (e.g. vitiligo, psoriasis) and controlled celiac disease;

4. treatment with anti-PD-1, anti-PD-L1, anti-PD-L2, or anti-CTLA-4 antibodies (or any other antibody that acts on the T cell costimulatory or checkpoint pathway);

5. uncontrolled hypertension (systolic pressure not less than 140mmHg and/or diastolic pressure not less than 90mmHg) or pulmonary hypertension or unstable angina; myocardial infarction or coronary artery bypass surgery and coronary artery stent implantation surgery are carried out within 6 months before administration; a history of chronic heart failure of grade 3-4 of the New York Heart Association (NYHA) criteria; valvular disease of clinical significance; severe arrhythmias requiring treatment, including QTc interval greater than 450ms for males and greater than 470ms for females (calculated by Fridericia equation); cerebrovascular accident (CVA) or Transient Ischemic Attack (TIA) within 12 months before administration;

6. patients with a history of arterial or deep venous thrombosis within 6 months prior to first study medication, or evidence or medical history of bleeding tendency within 2 months prior to first study medication, regardless of severity; partial prothrombin time (APTT) or Prothrombin Time (PT) > 1.5 × ULN;

7. skin wounds, surgical sites, trauma sites, severe ulceration of the mucosa or fractures do not heal completely;

8. the image shows evidence of tumor invasion into the large blood vessels, including the tumor having approached, surrounded, or invaded the lumen of the large blood vessels (e.g., pulmonary artery or superior vena cava);

9. dysphagia or known drug malabsorption;

10. researchers believe that gastrointestinal disorders that significantly affect oral drug absorption or conditions that may cause bleeding or perforation of the digestive tract (e.g., duodenal ulcers, ileus, acute crohn's disease, ulcerative colitis, large-area gastric and small bowel resections, etc.). Patients with chronic crohn's disease and ulcerative colitis (except total colon and rectal resection) should be excluded even in the inactive phase. Those suffering from hereditary nonpolyposis colorectal cancer or familial adenomatous polyposis syndrome. The patients with intestinal perforation and intestinal fistula history but not healed after the operation treatment;

11. present or previous development of interstitial pneumonia;

12. uncontrollable pleural and peritoneal cavities and pericardial effusion which need to be drained repeatedly or have obvious symptoms;

13. active infections requiring systemic treatment; active tuberculosis infection;

14. human immunodeficiency virus antibody (HIV-Ab) positive; active syphilis; hepatitis C antibody (HCV-Ab) positive and HCV-RNA > upper limit of normal for the unit of detection; hepatitis B surface antigen (HBsAg) positive and HBV-DNA copy number > upper limit of normal value of detection unit;

15. complications requiring treatment with immunosuppressive drugs, or complications requiring systemic or local administration of corticosteroids at immunosuppressive doses (prednisone >10 mg/day or equivalent doses of the same class of drugs);

16. live or attenuated vaccines are expected to be given 4 weeks before, during treatment or within 5 months of the last dose of study drug use;

17. inquired about the patients with drug taking history or drug abuse history;

18. lactating women (who gave consent to cessation of lactation during the trial period could be included);

19. other test drugs were treated or a trial instrument was used within 30 days before the study drug was started or within 5 half-lives (whichever is shorter) of the other test drugs;

20. known to be allergic to the study drug or any of its adjuvant components; a history of severe allergic diseases is known;

21. researchers believe it is not appropriate to participate in the clinical trial in other situations.

Test drug

Commercial furoquintinib tablets and anti-PD-1 antibody in example 1

Method of administration

Subjects received GB226 in combination with furazafenib treatment (specific dosing doses and methods see table 7) until either confirmed disease progression, intolerable toxicity, withdrawal of informed consent, initiation of other anti-tumor therapy, loss of visit, death, decision by the investigator subject to terminate therapy or withdraw from the study, or the end of the study.

TABLE 7 dosing regimen of GB226 combination furoquintinib

^*: if the first dose group appears to be intolerant of toxicity, a study by investigators and applicants may be conducted to reduce doses such as furoquintinib: 2mg and 1 mg/day for exploration;

#: if the second dose group is safe, the investigator and sponsor may increase one dose (furquintinib up to 5 mg/day) after discussion

Main end-points and evaluation indices:

incidence and characterization of adverse events, and changes in laboratory test results, physical examination, 12-lead electrocardiogram, and vital signs versus baseline

Dose Limiting Toxicity (DLT)

Maximum Tolerated Dose (MTD) or recommended extended dose (RDE)

Secondary endpoint and evaluation index:

evaluation indexes of clinical effectiveness include Objective Remission Rate (ORR), Disease Control Rate (DCR), duration of remission (DOR), progression-free survival (PFS), Overall Survival (OS)

Pharmacokinetic (PK) evaluation index, pharmacokinetic parameters of GB226 includeT_max，C_max，C_ss,min，R_C,troughEtc., the pharmacokinetic parameters of furoquintinib include T_max，C_max，AUC_0-24h，R_aucEtc. of

Immunogenicity of GB 226: number and percentage of subjects who produced anti-GB 226 antibody (ADA) and neutralizing antibody (NAb)

Exploratory endpoint and evaluation index:

expression level of PD-L1 and correlation of MSI and/or dMMR, TMB with therapeutic efficacy (ORR, DOR, DCR, PFS, OS)

Clinical results

First dose group safety summary

By 11 days 6 months 2019, all 3 subjects in the first dose group completed a 28 day DLT observation. During the DLT observation, no DLT event was observed. Subject dosing is shown in table 8.

TABLE 8 drug administration to subjects

In the first dose group 3 subjects experienced 1 adverse event of total bilirubin elevation within the 28-day DLT observation period, and 1 subject experienced no other adverse event.

The first dose group of this study was overall safe, 3 subjects did not develop DLT during the 28 day observation period. The pharmacokinetic profile of intravenous administration of GB 2263 mg/kg was not abnormal. By 12 months and 10 days 2019, the curative effect evaluation results of 3 patients in the first dose group are as follows: 1 case of PR (partial remission), 1 case of SD (stable disease), 1 case of IUPD (to confirm disease progression).

Example 3

Phase Ib with extended phase clinical trial of safety and efficacy of anti-PD-1 antibody in combination with furoquintinib in treatment of recurrent or metastatic non-small cell lung cancer (NSCLC) patients with EGFR-sensitive mutations who have failed Epidermal Growth Factor Receptor (EGFR) -Tyrosine Kinase Inhibitor (TKI) therapy

The experimental population is recurrent or metastatic NSCLC patients with EGFR-sensitive mutations who failed EGFR-TKI treatment.

The main purpose is as follows:

evaluation of the safety and tolerability of patients with recurrent or metastatic NSCLC of EGFR-sensitive mutations that failed treatment with GB226 combination Furosetinib for EGFR-TKI, determination of the Maximum Tolerated Dose (MTD) or recommended extended dose (RDE)

The secondary purpose is as follows:

Preliminary evaluation of the antitumor Activity of GB226 in combination with Fuquintinib for the treatment of recurrent or metastatic NSCLC of EGFR-sensitive mutations that failed EGFR-TKI treatment

Evaluation of the immunogenicity of the recurrent or metastatic NSCLC of the EGFR-sensitive mutation that has failed the treatment of EGFR-TKI with GB226 combination Furosetinib

The exploration purpose is as follows:

correlation of biomarkers such as programmed cell death protein ligand-1 (PD-L1) and microsatellite instability (MSI)/mismatch repair deficiency (dMMR), Tumor Mutational Burden (TMB) in tumor tissues of patients with recurrent EGFR-sensitive mutations or metastatic NSCLC who have failed EGFR-TKI treatment with the clinical efficacy of GB226 in combination with furoquintinib.

And (3) inclusion standard:

the test can be selected only if all the following conditions are met.

1.18-75 years old with unlimited character;

2. the test steps and contents are understood, written informed consent is voluntarily signed, the compliance is good, and follow-up visits are matched;

3. histologically or cytologically confirmed recurrent or metastatic NSCLC;

4. confirming that the EGFR gene sensitive mutation is positive, and according with one of the following: exon 19 deletion (19DEL), exon 21 point mutation (L858R/L861Q), 18 exon point mutation (G719X), 20 exon point mutation (S768I), and the following conditions were met:

detecting a subject without the T790M mutation after failure of EGFR-TKI treatment;

detecting a T790M mutant after failure of EGFR-TKI treatment, and failing to receive third-generation EGFR-TKI treatment; the primary T790M mutant, who progressed after receiving third-generation EGFR-TKI treatment or had no other effective treatment;

c. those who have failed chemotherapy or who are unwilling or intolerant to chemotherapy;

5. according to RECIST 1.1 criteria, a subject must have at least 1 measurable target lesion (lesion with a longest diameter ≧ 10mm, or lymph node with a short diameter ≧ 15 mm) examined by CT or MRI;

6. the expected life cycle is more than or equal to 3 months;

7, ECOG physical ability state score is 0-1 point;

8. pre-drug systemic chemotherapy, monoclonal antibody drug therapy, radical/broad radiotherapy, and prior anti-tumor biotherapy (tumor vaccine, cytokine or growth factor for tumor control) for at least 4 weeks; small molecule targeted drug therapy has been completed for at least 2 weeks; local palliative radiotherapy has been completed for at least 2 weeks;

9. EGFR-TKI treatment is over 2 weeks before the test medication;

10. has not received treatment with furoquintinib;

11. before the test drug administration, a major operation requiring general anesthesia must have been completed for at least 8 weeks; procedures requiring local/epidural anesthesia must have been completed for at least 4 weeks and the patient has recovered;

12. prior to trial administration, systemically administered corticosteroid drug (prednisone >10 mg/day or equivalent) has been discontinued for at least 2 weeks;

13. laboratory examinations made by screening must meet the following criteria:

blood routine (no blood transfusion, no use of G-CSF, no use of drug correction within 14 days prior to screening):

1) the hemoglobin HGB is more than or equal to 90 g/L;

2) absolute neutrophil count ANC ≥ 1.5X 10⁹/L；

3) Platelet PLT ≥ 100 × 10⁹/L；

Clinical biochemistry:

1) total Bilirubin (TBIL) is less than or equal to 1.5 times multiplied by the upper limit of normal value [ ULN ] (Gilbert syndrome allows less than or equal to 5 times multiplied by ULN);

2) aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) less than or equal to 2.5 times ULN (liver metastasis patients allow AST and/or ALT less than or equal to 5 times ULN);

3) serum creatinine (Cr) is less than or equal to 1.5 times multiplied by ULN or endogenous creatinine clearance rate is more than or equal to 50mL/min (Cockcroft-Gault formula);

4) the urine protein is less than 2+ or less than 1.0 g/L. For patients with urine protein more than or equal to 2+ or more than or equal to 1.0g/L at baseline, urine protein quantitative detection for 24 hours must be less than or equal to 1.0g/L for selection;

coagulation function:

1) activated Partial Thromboplastin Time (APTT) or Prothrombin Time (PT) is less than or equal to 1.5 times multiplied by ULN;

14. thyroid function index: thyroid Stimulating Hormone (TSH), free thyroxine (FT3/FT4) are in the normal range; if the TSH is not in the normal range, FT3/FT4 can be grouped in the normal range;

17. the subject needs to provide a tissue specimen and is willing to take a tissue biopsy when needed.

Exclusion criteria:

one who meets one of the following conditions will not be included in the test.

1. Squamous cell lung carcinoma patients (including adenosquamous carcinoma);

2. the gene detection result shows ALK fusion gene rearrangers;

3. patients who had previously suffered from other malignancies (except for cured cervical carcinoma in situ and basal cell carcinoma or squamous cell carcinoma) were not enrolled unless he/she had been cured at least 5 years prior to enrollment and were predicted to not require further anti-tumor treatment for the entire duration of the trial;

4. the imaging (CT or MRI) shows that the distance between the tumor focus and the large blood vessel is less than or equal to 5mm or the central tumor invading the local large blood vessel exists; or indicating the presence of a significant pulmonary cavitary or necrotic tumor;

5. active Central Nervous System (CNS) metastases, including symptomatic brain metastases or meningeal metastases or spinal cord compression, etc.; asymptomatic brain metastases can be enrolled (no progression for at least 4 weeks after radiation therapy and/or no neurological symptoms or signs after surgical resection, without treatment with glucocorticoids, anticonvulsants or mannitol);

6. symptomatic serosal cavity effusions such as peritoneal, pleural or pericardial effusions that are difficult to control;

7. patients with a history of arterial or deep vein thrombosis within 6 months prior to enrollment, or evidence or medical history of bleeding tendency within 2 months prior to enrollment, regardless of severity;

8. currently (within 10 days prior to the first use of the study drug) thrombolysis is being treated or therapeutic use of an anticoagulant (except prophylactic use);

9. there is an active, known history of autoimmune diseases, including but not limited to systemic lupus erythematosus, psoriasis, rheumatoid arthritis, inflammatory bowel disease, hashimoto's thyroiditis, and the like, with the exception of: type I diabetes, hypothyroidism which can be controlled by hormone replacement therapy alone, skin disorders which do not require systemic treatment (e.g. vitiligo, psoriasis) and controlled celiac disease.

10. Previously treated with an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, or an anti-CTLA-4 antibody (or any other antibody that acts on the T cell costimulatory or checkpoint pathway);

11. uncontrolled hypertension (systolic pressure not less than 140mmHg and/or diastolic pressure not less than 90mmHg) or pulmonary hypertension or unstable angina; myocardial infarction or bypass and stent operation are performed within 6 months before administration; a history of chronic heart failure meeting New York Heart Association (NYHA) criteria grade 3-4; severe arrhythmias requiring treatment, including QTc interval greater than 450ms for males and greater than 470ms for females (calculated by Fridericia equation); left Ventricular Ejection Fraction (LVEF) < 50%; cerebrovascular accident (CVA) or Transient Ischemic Attack (TIA) within 6 months before administration;

12. skin wounds, surgical sites, trauma sites, severe ulceration of the mucosa or fractures do not heal completely;

13. dysphagia or conditions where researchers believe gastrointestinal disturbances that significantly affect oral drug absorption or may cause bleeding or perforation of the digestive tract (e.g., duodenal ulcers, gastrointestinal obstruction, clinically active diverticulitis, intraperitoneal abscesses, peritoneal cancer metastasis, acute crohn's disease, ulcerative colitis, large area gastric and small bowel resection, etc.). Patients with chronic crohn's disease and ulcerative colitis (except total colon and rectal resection) should be excluded even in the inactive phase. Those suffering from hereditary nonpolyposis colorectal cancer or familial adenomatous polyposis syndrome. The patients with intestinal perforation and intestinal fistula history but not healed after the operation treatment;

14. those who have or have had an active tuberculosis infection or other active infections in need of systemic treatment;

15. human immunodeficiency virus antibody (HIV-Ab) positive; active syphilis; hepatitis C antibody (HCV-Ab) positive and HCV-RNA > upper limit of normal for the unit of detection; hepatitis B surface antigen (HBsAg) positive and HBV-DNA copy number > upper limit of normal value of detection unit;

16. complications requiring treatment with immunosuppressive drugs, or complications requiring systemic treatment at doses with immunosuppressive effects (prednisone >10 mg/day or congeneric drug equivalent doses), allowing for inhalation or topical use of steroid and prednisone or congeneric drug equivalent doses >10 mg/day in the absence of active autoimmune disease;

17. a history of interstitial lung disease;

18. the test drug is treated by other test drugs within 30 days before the test drug is used or within 5 half lives (whichever is shorter) of other test drugs or a test device is used within 30 days;

19. live or attenuated vaccines are expected to be administered within 4 weeks prior to administration, during treatment, or within 5 months of the last administration;

20. inquired about the patients with drug taking history or drug abuse history;

21. a lactating woman;

22. known to be allergic to recombinant humanized PD-1 mab or any adjuvant thereof; known to be allergic to furoquintinib analogues; known history of allergic disease or severe allergic constitution;

23. researchers have considered it inappropriate to participate in the clinical trial for various other reasons.

Test drug

GB226 injection:

the GB226 preparation is colorless to light yellow liquid with the specification of 70mg/7 ml/bottle, the GB226 is prepared in 100ml of 0.9% sodium chloride solution, the administration concentration needs to be controlled to be 1 mg/ml-10 mg/ml, the infusion is finished after the first administration for 60 minutes +/-10 minutes, and if no infusion reaction exists, the GB226 preparation can be adjusted to be more than or equal to 30 minutes later.

Fuquintinib capsule: two specifications, 1mg or 5mg respectively, are orally administered on an empty stomach once a day.

Method of administration

Dose escalation phase dosing:

primary end point:

frequency and characteristics of Adverse Events (AE) and Severe Adverse Events (SAE), and changes in laboratory tests, vital signs, electrocardiograms, and physical examinations, as compared to baseline

Dose Limiting Toxicity (DLT)

Maximum Tolerated Dose (MTD) or recommended extended dose (RDE)

Secondary endpoint:

the Pharmacokinetic (PK) evaluation index of GB226, including: peak concentration (C)_max) Time to peak (T)_max) Area under plasma concentration-time curve (AUC)_0-tAnd AUC_0-∞) Apparent volume of distribution (V)_d) Average residence time(MRT), total Clearance (CL); and the area under the plasma concentration-time curve (AUC) over the time interval between administrations_0-τ) Average steady state blood concentration (C)_avg) Minimum blood concentration (C)_min) And steady state Clearance (CL)_ss)

Blood drug concentration of furoquintinib

The efficacy evaluation indexes include: objective Remission Rate (ORR), Disease Control Rate (DCR), duration of remission (DOR), Progression Free Survival (PFS) and Total survival (OS)

And (3) exploring an end point:

programmed cell death protein ligand-1 (PD-L1) protein expression, microsatellite instability (MSI)/mismatch repair deficiency (dMMR), Tumor Mutation Burden (TMB) and efficacy (ORR, DOR, DCR, PFS, OS) correlation.

Clinical results

First dose group safety summary

By 2019, 9, 18 days, the first dose group of 3 subjects completed a 28-day DLT observation. During the DLT observation, no DLT event was observed. Subject dosing is shown in table 9.

TABLE 9 drug administration to subjects

The first dose group of the Ib stage accompanying expansion stage clinical test of the safety and effectiveness of the GB226 injection combined with furoquinib for treating the recurrent of EGFR sensitive mutation or metastatic non-small cell lung cancer patients who fail to treat EGFR-TKI has good overall safety, 3 subjects do not have DLT events in the DLT observation period, and the generated adverse events related to study medicaments are all 1 grade; severe adverse events were observed in 1 case of pleural effusion, due to disease progression. The occurrence of AE in combination is similar to the safety data of GB226 monoclonal antibody and furquintinib single drug, and the AE grade aggravation and new safety signals do not appear at present. The pharmacokinetics characteristics of 210mg intravenous administration of GB226 monoclonal antibody are not abnormal. By 12 months and 10 days 2019, the efficacy of 3 patients in the first dose group was evaluated as 1 SD (stable disease) and 2 PD (progressive disease).

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the equivalent replacement or change according to the technical solution and the modified concept of the present invention should be covered by the scope of the present invention.

SEQUENCE LISTING

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<120> use of anti-PD-1 antibody and furoquintinib in combination for preparing medicine for treating cancer

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Claims

1. Use of an anti-PD-1 antibody or antigen-binding fragment thereof and furoquintinib, or a pharmaceutically acceptable salt thereof, in combination for the manufacture of a medicament for the treatment of cancer, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises:

a light chain CDR1 sequence which is the amino acid sequence shown in SEQ ID NO. 1 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO. 1,

a light chain CDR2 sequence which is the amino acid sequence shown in SEQ ID NO. 2 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO. 2,

a light chain CDR3 sequence that is the amino acid sequence set forth in SEQ ID NO. 3, or that has at least 80% homology with the amino acid sequence set forth in SEQ ID NO. 3,

a heavy chain CDR1 sequence which is the amino acid sequence shown in SEQ ID NO. 4, or has at least 80% homology with the amino acid sequence shown in SEQ ID NO. 4,

a heavy chain CDR2 sequence which is the amino acid sequence shown in SEQ ID NO. 5, or has at least 80% homology with the amino acid sequence shown in SEQ ID NO. 5, and

a heavy chain CDR3 sequence which is the amino acid sequence shown in SEQ ID NO. 6, or has at least 80% homology with the amino acid sequence shown in SEQ ID NO. 6.

2. The use of claim 1, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

3. The use according to claim 2, wherein the humanized antibody or antigen-binding fragment thereof comprises a light chain variable region sequence which is the amino acid sequence set forth in SEQ ID NO. 7 or which has at least 80% homology with the amino acid sequence set forth in SEQ ID NO. 7.

4. The use according to claim 2, wherein the humanized antibody or antigen binding fragment thereof comprises a heavy chain variable region sequence which is the amino acid sequence set forth in SEQ ID NO. 8 or which has at least 80% homology with the amino acid sequence set forth in SEQ ID NO. 8.

5. The use of claim 2, wherein the humanized antibody heavy chain sequence is as set forth in SEQ ID NO 9 and the light chain sequence is as set forth in SEQ ID NO 10.

6. The use of claim 1, wherein the cancer expresses PD-L1.

7. The use of claim 1, wherein the cancer is lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, intestinal cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, stomach cancer, testicular cancer, salivary gland cancer, thyroid cancer, thymus cancer, epithelial cancer, head and neck cancer, stomach cancer, pancreatic cancer, prostate cancer, kidney cancer, or a combination thereof.

8. The use of claim 1, wherein the cancer is non-small cell lung cancer and colon cancer.

9. The use according to claim 1, wherein the anti-PD-1 antibody is used in an amount of 0.1-40mg/kg or a fixed dose of 100-400 mg.

10. The use according to claim 9, wherein the anti-PD-1 antibody is used in an amount of 3mg/kg or a fixed dose of 210 mg.

11. The use of any one of claims 9-10, wherein the anti-PD-1 antibody is administered biweekly.

12. The use according to claim 9, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is used in an amount of 5 mg/kg.

13. The use of claim 12, wherein the anti-PD-1 antibody is administered twice weekly.

14. The use according to claim 1, wherein the furoquintinib is used in an amount of 0.1-10mg/kg or in a fixed dose of 0.1-20 mg.

15. The use according to claim 14, wherein the amount of furoquintinib is 1mg/kg, 2mg/kg, or a fixed dose of 2mg, 3mg, 4mg or 5mg is used.

16. The use according to any one of claims 14 to 15, wherein the furoquintinib is administered once daily.

17. The use of claim 16, wherein the furoquintinib is administered for three weeks, discontinued for one week.

18. The use of any one of the preceding claims, wherein the anti-PD-1 antibody or antigen-binding fragment thereof and furoquintinib, or a pharmaceutically acceptable salt thereof, are administered during the same dosing cycle.

19. A kit comprising the anti-PD-1 antibody of any one of claims 1-5 and furoquintinib, or a pharmaceutically acceptable salt thereof.