WO2021143799A1 - Use of anti-pd-1 antibody in combination with fruquintinib in preparation of medicaments for treating cancer - Google Patents

Abstract

Use of an anti-PD-1 antibody or an antigen binding fragment thereof in combination with fruquintinib or a pharmaceutically acceptable salt thereof in the preparation of medicaments for treating cancer. The combined administration of the anti-PD-1 antibody and fruquintinib has a certain synergistic effect. Compared with the corresponding single administration, the effect of inhibiting tumor growth is obvious. During administration, animals for testing have good tolerance and no adverse reactions.

Description

Application of anti-PD-1 antibody and fruquintinib in the preparation of drugs for treating cancer

This application claims the priority of Chinese Patent Application No. 202010052280.6 filed on January 17, 2020.

Technical field

The present invention relates to the field of medicine, and more specifically, to the use of an anti-PD-1 antibody or an antigen-binding fragment thereof and fruquintinib or a pharmaceutically acceptable salt thereof in the preparation of a medicine for treating cancer.

Background technique

Vascular endothelial cell growth factor (vascular endothelial cell growth factor, VEGF) is currently found to be one of the main inducers related to tumor angiogenesis. Combining with VEGF receptor (VEGFR) can activate angiogenesis, leading to continuous tumor growth and metastasis diffusion. Blocking the VEGF/VEGFR signal transduction pathway can inhibit angiogenesis, thereby inhibiting tumor growth and achieving anti-tumor effects. At present, a number of VEGF/VEGFR inhibitors have been approved, including monoclonal antibody drug bevacizumab and small molecule kinase inhibitor drugs sunitinib, sorafenib, lenvatinib, regofini, and A Xitinib, Fruquintinib and so on.

Patent number CN101575333B discloses the VEGFR small molecule inhibitor Fruquintinib (Fruquintinib), the chemical name is 6-(6,7-dimethoxyquinazoline-4-oxo)-N,2-dimethylbenzene Difuran-3-carboxamide, the molecular formula is C ₂₁ H ₁₉ N ₃ O ₅ , and its structural formula is shown in the following formula:

Fruquintinib is a highly selective tumor angiogenesis inhibitor. Its main targets are VEGFR kinase family VEGFR1, VEGFR2 and VEGFR3. It can inhibit VEGFR phosphorylation, thereby inhibiting tumor angiogenesis, and ultimately inhibiting tumor growth. .

Programmed death receptors (Programmed Death-1, PD-1) and its ligands PD-L1/L2 are important T-cell negative regulatory immune checkpoints, which regulate the body’s peripheral immune tolerance, which is important for peripheral immunity. Balance plays an important role. Cancer cells can inhibit immune cell activation by highly expressing PD-L1 and binding to the PD-1 receptor on the surface of T-cells, and induce tumor immune tolerance. PD-1/PD-L1 plays a role in tumor immune escape and tumor microenvironment formation. To an important role. The monoclonal antibody drugs represented by targeting PD-1 and its ligand PD-L1 block the PD1/PD-L1 negative immune check site to activate and proliferate T-cells, which can reverse the tumor immunosuppressive microenvironment and enhance resistance Tumor immune response to achieve tumor immunotherapy, thereby effectively inhibiting tumor growth. The anti-PD-1 antibody of Jiahe Biopharmaceutical Industry (CN106573052A discloses the antibody and its preparation method, also referred to as GB226 hereinafter), is currently in clinical phase II, has good safety, and has effective anti-tumor effects.

In the process of cancer treatment, the therapeutic effect of single administration has certain limitations, the drug effect is not obvious, and drug resistance is easy to develop. For example, patients with metastatic colorectal cancer and EGFR-TKI-resistant non-small cell lung cancer who have failed standard treatments are currently treated with very limited treatment options. New treatment methods and drugs are urgently needed to improve the efficacy and prolong the survival of patients.

At present, clinical studies on the combination of anti-PD-1 antibodies and VEGFR inhibitors in the treatment of cancer are underway, but there is no information on approval 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 an anti-PD-1 antibody Use in combination with axitinib.

Summary of the invention

The present invention provides the use of an anti-PD-1 antibody or an antigen-binding fragment thereof and fruquintinib or a pharmaceutically acceptable salt thereof in the preparation of a medicine for treating cancer.

Preferably, the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof comprises a light chain CDR1 (LCDR1) sequence, which is an amino acid sequence as shown in SEQ ID NO:1, or is similar to the sequence shown in SEQ ID NO:1 The amino acid sequence has at least 80% homology, the light chain CDR2 (LCDR2) sequence, which is the amino acid sequence shown in SEQ ID NO: 2, or has at least 80% of the amino acid sequence shown in SEQ ID NO: 2 The 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. 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, 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 the heavy chain CDR3 (HCDR3) sequence, which is shown in SEQ The amino acid sequence shown in ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 6 has at least 80% homology.

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

Preferably, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a light chain variable region sequence, which is an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence as shown in SEQ ID NO: 7 The sequence has at least 80% homology.

Preferably, the aforementioned anti-PD-1 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 is the same as the amino acid sequence shown in SEQ ID NO: 8 Have at least 80% homology.

Preferably, the heavy chain sequence of the aforementioned anti-PD-1 antibody is the sequence shown in SEQ ID NO: 9, and the light chain sequence is the sequence shown in SEQ ID NO: 10.

Preferably, the aforementioned cancer has one or more of the following characteristics:

Express PD-L1, high microsatellite instability (MSI-H), mismatch repair defect (dMMR) and high tumor mutational burden (TMB).

Preferably, the above-mentioned cancer is lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, bowel 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, pancreatic cancer, prostate cancer, or kidney cancer.

Preferably, the above-mentioned bowel cancer is colon cancer, rectal cancer, large intestine cancer, small intestine cancer or colorectal cancer.

More preferably, the above-mentioned bowel cancer is colorectal cancer.

Preferably, the aforementioned colorectal cancer is metastatic colorectal cancer.

Preferably, the above-mentioned colorectal cancer is microsatellite stable (MSS) and/or mismatch repair function intact or high degree of microsatellite instability and/or mismatch repair defect.

Preferably, the aforementioned lung cancer is non-small cell lung cancer.

Preferably, the aforementioned non-small cell lung cancer is recurrent or metastatic non-small cell lung cancer with EGFR sensitive mutations.

Preferably, the recurrence or metastatic non-small cell lung cancer of the above-mentioned EGFR-sensitive mutation of the EGFR-sensitive mutation is the failure of EGFR-TKI treatment.

Preferably, the aforementioned non-small cell lung cancer has one or more of the following characteristics:

Exon 19 deletion (19DEL), exon 21 point mutation (L858R/L861Q), exon 18 point mutation (G719X) and exon 20 point mutation (S768I).

Preferably, the single administration dose of the aforementioned anti-PD-1 antibody is 0.1-40 mg/kg individual body weight or a single fixed dose of 100-400 mg.

More preferably, the single administration dose of the above-mentioned anti-PD-1 antibody is 3 mg/kg individual body weight or a single fixed dose of 210 mg.

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

Preferably, the single administration dose of the above-mentioned anti-PD-1 antibody is 5 mg/kg individual body weight. Preferably, it is administered twice a week.

More preferably, the above-mentioned anti-PD-1 antibody is GB226, and its dosage is 3 mg/kg administered according to the patient's body weight or a fixed dose of 210 mg, administered once every two weeks.

Preferably, the single administration dose of Fruquintinib is 0.1-10 mg/kg individual body weight or a single fixed dose of 0.1-20 mg.

More preferably, the single administration dose of Fruquintinib is 1 mg/kg, 2 mg/kg of individual body weight, or a single fixed dose of 1 mg, 2 mg, 3 mg, 4 mg or 5 mg.

More preferably, Fruquintinib is administered in a single fixed dose of 2 mg, 3 mg, 4 mg or 5 mg.

More preferably, Fruquintinib is administered once a day. More preferably, the drug is administered for three weeks and the drug is stopped for one week, or the drug is administered for two weeks and the drug is stopped for one week.

More preferably, a single fixed dose of 2 mg of Fruquintinib is administered once a day for three weeks and the drug is stopped for one week.

More preferably, a single fixed dose of 3 mg of Fruquintinib is administered once a day for three weeks and the drug is stopped for one week.

More preferably, a single fixed dose of Fruquintinib is 4 mg, which is administered once a day for three weeks, and the drug is stopped for one week.

More preferably, a single fixed dose of Fruquintinib is administered at 5 mg, once a day for three weeks, and the drug is stopped for one week. Preferably, the anti-PD-1 antibody or its antigen-binding fragment and fruquintinib or its pharmaceutically acceptable salt are administered in the same administration cycle.

Preferably, in the use of the present invention, when the above-mentioned colorectal cancer is metastatic colorectal cancer, the single administration dose of PD-1 antibody or antigen-binding fragment thereof is 3 mg/kg individual body weight, and the administration frequency is every two weeks. The drug is administered once; and a single fixed dose of Fruquintinib is 3 mg, the frequency of administration is once a day for three consecutive weeks, and the drug is stopped for one week.

Preferably, in the use of the present invention, when the above-mentioned colorectal cancer is metastatic colorectal cancer, the single administration dose of the anti-PD-1 antibody or antigen-binding fragment thereof is 3 mg/kg individual body weight, and the administration frequency is It is administered once every two weeks; and the single fixed dose of Fruquintinib is 4 mg, and the frequency of administration is once a day for three consecutive weeks, and the drug is stopped for one week.

Preferably, in the use of the present invention, when the aforementioned lung cancer is recurrent or metastatic non-small cell lung cancer with EGFR-sensitive mutations that have failed EGFR-TKI treatment, the fixed dose of anti-PD-1 antibody or its antigen-binding fragment is 210 mg for a single administration , The frequency of administration is once every two weeks; and a single fixed dose of fruquintinib is 5 mg, and the frequency of administration is once a day for three consecutive weeks, and the drug is stopped for one week.

The present invention also provides a kit containing the above-mentioned anti-PD-1 antibody or antigen-binding fragment thereof and fruquintinib or a pharmaceutically acceptable salt thereof.

The present invention also provides an anti-PD-1 antibody or an antigen-binding fragment thereof combined with fruquintinib or a pharmaceutically acceptable salt thereof as a therapeutic preparation for treating tumors.

The present invention also provides the above-mentioned anti-PD-1 antibody or its antigen-binding fragment in combination with fruquintinib or its pharmaceutically acceptable salt as a medicine for reducing adverse drug reactions, the adverse reactions of the above-mentioned drugs are selected from those caused by anti-PD-1 antibodies or Caused by Fruquintinib or a pharmaceutically acceptable salt thereof.

The present invention also provides the above-mentioned anti-PD-1 antibody or its antigen-binding fragment in combination with fruquintinib or a pharmaceutically acceptable salt thereof as a single administration dose reduction of anti-PD-1 antibody and/or fruquintinib or its pharmaceutically acceptable salt. The received salt is administered with a dose of drug alone.

The present invention also provides a method for reducing the dose of anti-PD-1 antibody or its antigen-binding fragment alone and/or the dose of Fruquintinib or its pharmaceutically acceptable salt alone, comprising administering the above-mentioned anti-PD-1 antibody to a patient. PD-1 antibody combined with Fruquintinib or a pharmaceutically acceptable salt thereof.

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

The present invention also provides a kit containing the above-mentioned anti-PD-1 antibody or antigen-binding fragment thereof and fruquintinib or a pharmaceutically acceptable salt thereof.

Description of the drawings

Figure 1 shows the growth curve of a mouse colorectal cancer MC38 model after the start of treatment. This figure illustrates the effect of the anti-PD-1 antibody of the present invention and furquintinib alone or in combination on the tumor volume of the mouse colorectal cancer MC38 model It can be seen from the figure that at the end of the control group (PG-D17), the tumor growth inhibition rate of the GB226 group, Fruquintinib high-dose group, Fruquintinib low-dose group, GB226+Fruquintinib high-dose group, and GB226+Fruquintinib low-dose group They were 90%, 73%, 56%, 94%, 93%. The tumor volume of each treatment group was significantly lower than that of the control group (p<0.05). Before the end of the experiment (PG-D24), the tumor volume of the GB226+Fruquintinib high-dose group and the GB226+Fruquintinib low-dose group were significantly lower than the corresponding GB226 and Fruquintinib single-dose groups (p<0.05), and there was no significant difference between the combination groups ( p=0.885).

Figure 2 shows the weight change curve of experimental animals after the start of treatment. This figure illustrates the weight change curve of experimental animals after the anti-PD-1 antibody of the present invention and fruquintinib are administered alone or in combination. It can be seen from the figure during the treatment period The tumor-bearing mice showed good tolerance to the test drugs Fruquintinib, GB226 and their combination. The mice in each group had normal body weight, no abnormal performance, and were generally in good condition.

Figure 3 shows the percentage of weight change of experimental animals after the start of treatment, which illustrates the percentage of weight change of experimental animals after the anti-PD-1 antibody of the present invention and fruquintinib are administered alone or in combination. It can be seen from the figure that during the treatment period, the tumor-bearing mice showed good tolerance to the test drugs Fruquintinib, GB226 and their combination. The mice in each group had normal body weight, no abnormal performance, and were generally in good condition.

Detailed ways

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

Table 1

name	serial number	sequence
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 description of the present invention, the light chain and heavy chain variable region sequences of the above-mentioned humanized antibodies are shown in Table 2 below.

Table 2

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

table 3

In the use described in the present invention, the above-mentioned cancer is a cancer that expresses PD-L1. The aforementioned cancers include, but are not limited to, lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, bowel cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, liver 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. In a preferred embodiment of the present invention, the above-mentioned colon cancer is colorectal cancer. In a preferred embodiment of the present invention, the aforementioned colorectal cancer is metastatic colorectal cancer. In a preferred embodiment of the present invention, the aforementioned lung cancer is non-small cell lung cancer (NSCLC). In a more preferred embodiment of the present invention, the above-mentioned non-small cell lung cancer is epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) treatment failure of EGFR sensitive mutation recurrence or metastatic non-small cell lung cancer (NSCLC).

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. For the purpose of the present invention, the following terms are defined below.

When the term "and/or" is used to connect two or more alternatives, it should be understood to mean any one of the alternatives or any two or more of the alternatives.

As used herein, the term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In this document, when the term "comprises" or "includes" is used, unless otherwise specified, it also encompasses the situation consisting of the stated elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a specific sequence, it is also intended to encompass the antibody variable region composed of that specific sequence.

The term "antibody" is used in the broadest sense and refers to a protein containing an antigen-binding site, covering natural and artificial antibodies of various structures, including but not limited to intact antibodies and antigen-binding fragments of antibodies.

The term "dose" is the amount of a drug that induces a therapeutic effect. Unless otherwise stated, the dosage is related to the amount of the free form of the drug. If the drug is in the form of a pharmaceutically acceptable salt, the amount of the drug is increased in proportion to the amount of the drug in the free form. For example, the dosage will be stated on the product packaging or product information sheet.

The term "administration" refers to the physical introduction of each active ingredient of the drug of the present invention into an individual using any of a variety of methods and delivery systems known to those skilled in the art. The route of administration of each active ingredient in the medicine of the present invention includes oral, intravenous (e.g., infusion (also known as drip) or injection), intramuscular, subcutaneous, intraperitoneal, spinal, local or other parenteral administration way. As used herein, the phrase "parenteral administration" refers to methods of administration other than gastrointestinal and topical administration, usually via intravenous, and without limitation includes intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intrasaccular , Intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspine, epidural and intrasternal injection and infusion, and in vivo electroporation. Correspondingly, each active ingredient in the medicine of the present invention can be formulated into capsules, tablets, injections (including infusions or injections), syrups, sprays, lozenges, liposomes or suppositories, etc.

The term "continuous administration" refers to daily administration. In the case of continuous administration, the drug may be administered one or more times a day, for example, the drug may be administered at a frequency of once a day, twice a day, or three times a day, preferably at a frequency of once a day.

The term "treatment" includes administering the drug of the present invention to an individual in need to achieve the purpose of curing the disease or having an effect on the regression of the disease or delaying the progression of the disease. When referring to a disease, the term "treatment" refers to alleviating the disease (ie, slowing down or preventing or reducing the development of the disease or at least one clinical symptom thereof), preventing or delaying the onset or development or progression of the disease.

The term "pharmaceutically acceptable salt" refers to a salt of fruquintinib that is non-toxic, biologically tolerable, or otherwise biologically suitable for the administration of fruquintinib for the treatment or prevention of diseases. Including but not limited to acid addition salt or base addition salt, for example: acid addition salt formed by furquintinib and inorganic acid, such as hydrochloride, hydrobromide, carbonate, bicarbonate, phosphate , Sulfates, sulfites, nitrates, etc.; and acid addition salts formed by furquintinib and organic acids, such as formate, acetate, malate, maleate, fumarate, Tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, benzoate, salicylate, stearate and The salt of alkane dicarboxylic acid of the formula HOOC-(CH ₂ ) _n -COOH (where n is 0-4), etc.

In the use described in the present invention, the single administration dose of anti-PD-1 antibody may be 0.1-40 mg/kg, preferably 0.5-20 mg/kg, more preferably 1-10 mg/kg, most preferably 3 mg/kg or 5 mg/kg; For adults, a fixed dose may also be used, such as 100-400 mg, preferably 150-300 mg, and 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 present invention, the single administration dose of Fruquintinib may be 0.1-10 mg/kg, preferably 0.5-5 mg/kg. For adults, a fixed dose may be used, for example 0.1-20 mg, preferably 1-10 mg, more preferably 1 mg, 2 mg, 3 mg, 4 mg or 5 mg. The frequency of administration of Fruquintinib can be once a day, more preferably three weeks of continuous administration, and one week of withdrawal.

In the application described in the present invention, the anti-PD-1 antibody is preferably administered by injection, such as subcutaneous or intravenous injection, and the anti-PD-1 antibody needs to be formulated into an injectable form before injection. The injectable form of the anti-PD-1 antibody may be an injection or a lyophilized powder injection, which contains an anti-PD-1 antibody, a buffer, a stabilizer, and optionally a surfactant. The buffer can be selected from one or more of 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 esters, polyoxyethylene sorbitan fatty acid esters, preferably the above-mentioned polyoxyethylene sorbitan fatty acid esters are polysorbate 20, 40, 60 or 80, Polysorbate 20 is most preferred. The most preferred injectable form of anti-PD-1 antibody contains anti-PD-1 antibody, acetate buffer, trehalose and polysorbate 20. Fruquintinib is preferably administered orally, for example, it can be formulated into tablets or capsules for administration.

In the use described in the present invention, "combination" is a mode of administration, which includes various situations in which two drugs are administered sequentially or simultaneously. The so-called "simultaneous" herein refers to the administration of anti-PD- in the same administration cycle. 1 Antibody and Fruquintinib, for example, two drugs are given within 2 days or 1 day. The so-called "sequential" administration includes the administration of anti-PD-1 antibody and Fruquintinib in different administration cycles. These administration modes all belong to the above-mentioned combined administration of the present invention.

In the application described in the present invention, when the anti-PD-1 antibody is used in combination with fruquintinib, it can reduce the adverse drug reaction caused by the anti-PD-1 antibody and/or fruquintinib. The aforementioned adverse reactions can be selected from vascular-related adverse reactions, glandular hypofunction, skin adverse reactions, respiratory system adverse reactions, liver-related adverse reactions, endocrine-related adverse reactions, digestive system adverse reactions, kidney-related adverse reactions, fatigue, fever; The aforementioned vascular-related adverse reactions can be selected from hemangioma, vasculitis, lymphangioma, and the aforementioned hypothyroidism can be selected from hypothyroidism, hypoparathyroidism, hypopancreatic hypofunction, and prostate hypofunction; the aforementioned skin adverse reactions are optional Self-pruritus, urticaria, skin rash, toxic epidermal necrosis; the above-mentioned respiratory system adverse reaction is selected from pneumonia, bronchitis, chronic obstructive pulmonary disease, pulmonary fibrosis; the above-mentioned liver-related adverse reaction may be selected from hepatitis, abnormal liver function; Endocrine-related adverse reactions can be selected from type I diabetes, type II diabetes, and hypoglycemia; kidney-related adverse reactions can be selected from nephritis, renal failure; the above-mentioned digestive system adverse reactions can be selected from diarrhea, nausea, vomiting, enteritis, and constipation. More preferably, the aforementioned adverse drug reactions may be selected from hemangioma, hypothyroidism, and parathyroid function.

In the method for reducing the dose of anti-PD-1 antibody alone and/or the dose of fruquintinib alone described in the present invention, when used in combination with the above-mentioned anti-PD-1, the dose of fruquintinib is that of the dose alone. 10%-100% of the dose, preferably 10%-75%, more preferably 75%, 50%, 25%, 12.5%. When combined with Fruquintinib, the dosage of the anti-PD-1 antibody is 10%-100% of the dosage of the anti-PD-1 antibody alone, preferably 10%-50%.

The term "microsatellite (MS)" refers to a DNA sequence repeated in a few nucleotides (mostly 1-6) in the cell genome, also known as short tandem repeats. When the DNA mismatch repair (MMR) function is abnormal, the replication errors of MS cannot be corrected and continue to accumulate, which changes the sequence length and base composition of MS and becomes microsatellite instablity (MSI) At the same time, the genome exhibits a highly mutational phenotype. MSI is divided into MS highly unstable (MSI-H), MS low unstable (MSI-L) and MS stable (MSS). The incidence of MSI-H in colorectal cancer is 12-15%, of which stage IV colorectal cancer is 4-5%.

MSI is mostly caused by MMR functional defects caused by the lack of MMR protein expression, so the MSI state can be reflected by detecting the lack of MMR protein. Detection of MMR protein expression and detection of MSI status based on DNA analysis are different methods to evaluate the same biological effects. One method to detect the expression of MMR protein is the IHC method, which uses specific antibodies against MLH1, MSH2, MSH6, and PMS2, respectively, and locates the nucleus to check the positive expression. If all four MMR proteins are positively expressed in the tumor sample, it means that the MMR function is intact (proficient mismatch repair, pMMR); the absence of any MMR protein is dMMR. Generally speaking, dMMR is equivalent to MSI-H, and pMMR is equivalent to MSI-L or MSS phenotype. In this application, "highly microsatellite instability (MSI-H)/mismatch repair defect (dMMR)", "highly microsatellite instability (MSI-H) and/or mismatch repair defect (dMMR)" and " MSI-H/dMMR" means the same meaning and can be interchanged. "Microsatellite stabilization (MSS) / complete mismatch repair function (pMMR)", "microsatellite stabilization (MSS) and/or complete mismatch repair function (pMMR)" have the same meaning as "MSS/pMMR" and can be interchanged.

The term "tumor mutation burden (TMB)" is the total number of mutations in a tumor specimen. TMB has been proven to be independent of MSI status and PD-L1 expression as a predictor of efficacy in multiple solid tumors. It is currently believed that the higher the TMB, the stronger the tumor immunogenicity, the greater the probability that tumor cells are recognized by T cells, and the use of immunosuppressive agents is more likely to cause an immune response. In colorectal cancer, dMMR tumor TMB is significantly higher than pMMR tumor. Therefore, the present invention uses TMB as an exploratory index in the research of colorectal cancer, and intends to further explore the relationship between TMB and the curative effect of advanced colorectal cancer.

In this application, terms such as “first-line treatment”, “second-line treatment” and “third-line treatment” refer to the treatment methods adopted for tumor patients clinically, and the first-line treatment refers to the intervention measures taken for the first treatment of the patient’s condition , Including surgery and chemotherapy drugs. The second-line mainly refers to the failure of the first treatment, or after a period of time after the first treatment, such as chemotherapy patients after six to eight times of chemotherapy, which may cause resistance to chemotherapy drugs, which will not have a therapeutic effect on tumor treatment. At this time, second-line treatment options can be considered. For patients who have failed second-line treatment, that is, patients who still have disease progression after treatment, consider a third-line treatment plan. Usually, the anti-cancer mechanisms used in different number of treatments are different.

In this application, "EGFR-TKI treatment" refers to a treatment regimen that uses a tyrosine kinase inhibitor (TKI) targeting epidermal growth factor receptor (EGFR). Common EGFR-TKI drugs include gefitinib, erlotinib, icotinib, afatinib and so on. Epidermal growth factor receptor (EGFR) is overexpressed, dysregulated or mutated in many epithelial malignancies, and EGFR activation is important in tumor growth and progression. As an important target for non-small cell lung cancer (NSCLC) targeted therapy, EGFR has a high frequency of mutations in Asian populations. EGFR-TKI has achieved good results in the treatment of non-small cell lung cancer. And accumulated a lot of clinical experience. However, most patients who responded to EGFR-TKI treatment developed drug resistance after receiving EGFR-TKI treatment. How to delay the occurrence of EGFR TKIs resistance, optimize the treatment plan so as to maximize the efficacy of first-line treatment and prolong survival is always a problem of clinical concern.

In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention will be further explained in conjunction with the embodiments below.

Example 1

The in vivo pharmacodynamic study of Fruquintinib combined with anti-PD-1 antibody on PD-1 humanized MC38 mouse tumor model.

1. Raw material information and sources

1.1 Raw material information

1.1.1 Fruquintinib: The chemical name is 6-(6,7-dimethoxyquinazoline-4-oxo)-N,2-dimethylbenzofuran-3-carboxamide , The molecular formula is C ₂₁ H ₁₉ N ₃ O ₅ , and its preparation method is disclosed in the CN101575333B patent. The structural formula is as follows:

1.1.2 GB226 sequence information: The anti-PD-1 antibody is prepared according to the method disclosed in CN106573052A, and the heavy and light chain sequences of the anti-PD-1 antibody are shown in SEQID NO: 9 and SEQID NO: 10 of the present invention.

1.2 Source of raw materials

1.2.1 Fruquintinib:

Provided by: Yikang (Beijing) Pharmaceutical Technology Co., Ltd.

Supplier: He Quan Pharmaceutical Co., Ltd.

Batch: C13052210-AF18003M

Shape: white powder

Quantity: 50mg/bag

1.2.2 Anti-PD-1 antibody:

Provided by: Jiahe Biological Pharmaceutical Co., Ltd.

Batch: RD-201805001

Properties: transparent liquid

Quantity: 70mg/bottle, 3 bottles

2. Raw material configuration method and storage conditions

Anti-PD-1 antibody (10mg/ml): Use a micropipette to accurately measure 0.3ml sample stock solution into a glass bottle, take another 5.7ml PBS to the glass bottle and mix thoroughly to a final concentration of 0.5mg/ml, for use now Now equipped

Fruquintinib: accurately weigh 5.6 mg of fruquintinib sample into a glass bottle, measure 28 ml of 0.5% CMC-Na into the glass bottle, vortex for 1-3 minutes and sonicate with an ultrasonic cleaner for 15 minutes , Mix thoroughly to the final concentration of 0.2mg/ml, prepare once a week and sub-package, store at 4℃;

3. Laboratory animals and feeding management

3.1. Experimental animals

Mus Musculus, huPD-1 C57BL/6 (mouse, PD-1), 6-8 weeks old, all females, weighing 18-23g, a total of 62 (enrolled in 48); the experimental animal was made by Beijing Weitong Provided by Lihua Laboratory Animal Technology Co., Ltd., production license number: SCXK (Beijing) 2016-0011, quality certificate number: 1100111911048361.

3.2 Feeding management

The experimental animals were kept in a clean room with SPF-grade constant temperature and humidity in a laminar flow, using independent ventilated cages with IVC, one cage for every 4 rats, and the litter changed twice a week. Each cage has a cage label indicating the number of animals , Gender, strain, receiving time, group and start time of experiment. The temperature and humidity are controlled in the range of (23±3)℃/40-70%. SPF rodent feed, sterilized by cobalt 60 irradiation, drinking water is ultrafiltration purified water, and after autoclaving, animals can freely ingest sterile food and drinking water. Animals are numbered by ear punching.

4. Experimental method

4.1 Cell culture

MC38 mouse colon cancer cells (YK-CL-256-02) were purchased from Biovector NTCC Inc. (Biovector NTCC Inc.), with inactivated 10% fetal bovine serum, 100U/ml penicillin Cultivate tumor cells in a DEME medium with 100μg/ml streptomycin and 2mM glutamine in a 37°C, 5% CO ₂ incubator. After the cells are overgrown every 3 to 4 days, the tumor cells will be subcultured. Tumor cells in logarithmic growth phase are used for inoculation of tumors in vivo.

4.2 Tumor cell inoculation and grouping

MC38 tumor cells resuspended in PBS, at a concentration of 1×10 ⁷ /ml, were inoculated into the right anterior flank subcutaneously of PD-1 humanized C57BL/6 mice, 100 μl/mouse, and grouped when the ^{tumor grew to about 88 m 3} Dosing (recorded as PG-D0 on the day), a total of 6 groups, 8 in each group, the specific dosing schedule is shown in Table 4:

Table 4. Dosing schedule

Note: *The dosage volume is 10μl/g based on the animal's body weight, and the dosage can be adjusted when the body weight drops by 15-20%;

ip: intraperitoneal injection; po: intragastric administration; biw×4: administration twice a week for a total of 4 administrations; qd×18/23/26: administration once a day for a total of 18, 23, 26 days (times).

4.3 Measurement of mouse body weight and experimental indicators

A vernier caliper was used to measure the tumor volume three times a week, and the mice were weighed with an electronic balance to measure the long and short diameters of the tumor. The volume calculation formula was: volume = 0.5×long diameter×short diameter ² . See Table 5 for the measurement results. The T/C value was calculated according to the tumor volume, where T is the average value of Relative Tumor Volume (RTV) of each test substance treatment group, and C is the average value of Relative Tumor Volume (RTV) of the control group. The T/C value is the ratio of tumor volume after administration to before administration. Tumor growth inhibition rate (%)=(1-T/C)×100%. During the experiment, in order to meet the ethical norms of experimental animals, the mice were euthanized ^{if the tumor volume was greater than 2000 mm 3.} Therefore, the mice in the control group were sacrificed at PG-D17; the mice in the low-dose group of Fruquintinib were sacrificed at PG-D22; the GB226 group, the high-dose Fruquintinib group, the GB226+Fruquintinib high-dose group, and the GB226+Furaquinib All mice in the quintinib low-dose group were killed at PG-D25. After the mice were euthanized, the tumors were taken and weighed. Since the weight of the tumor can only be weighed after the mouse is sacrificed, the weight of the tumor in each administration group and the control group is only measured once. The tumor weight inhibition rate calculation formula is 1-the ratio of the tumor weight between the administration group and the control group. The tumor weight in the control group is based on the tumor weight at PG-D17, and the tumor weight in the low-dose fruquintinib group is based on PG-D22 , Other groups are subject to PG-D25 tumor weight. See Table 6 for the measurement results.

5. Experimental results

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 Figure 1) and the tumor weight (see Table 6), the results show that when the control group ends (PG-D17), GB226 The tumor growth inhibition rates of the Fruquintinib group, Fruquintinib high-dose group, Fruquintinib low-dose group, GB226+Fruquintinib high-dose group, and GB226+Fruquintinib low-dose group were 90%, 73%, 56%, 94%, 93%, respectively. The tumor volume was significantly lower than the control group (p<0.05). Before the end of the experiment (PG-D24), the tumor volume of the GB226+Fruquintinib high-dose group and the GB226+Fruquintinib low-dose group were significantly lower than the corresponding GB226 and Fruquintinib single-dose groups (p<0.05), and there was no significant difference between the combination groups ( p=0.885).

Tumor weight changes (see Table 6): mouse tumor weight in GB226 group, Fruquintinib high-dose group, Fruquintinib low-dose group, GB226+Fruquintinib high-dose group, GB226+Fruquintinib low-dose group, tumor weight inhibition rates were 40.1%, 19.35, respectively %, 10.57%, 83.33%, 83.30%. Through the inhibition rate of tumor weight, it can be found that the combination medication has a significant synergistic effect.

Table 6. Tumor inhibitory effect of test substance on mouse MC38 model of colorectal cancer (tumor weight)

Note: ^a. Mean ± standard error.

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

5.2 Mice safety impact

The body weight changes of experimental animals after the start of treatment (see Figure 2 and Figure 3). The results showed that during the treatment period, the tumor-bearing mice showed good tolerance to the test substances Fruquintinib, GB226 and their combination. The mice in each group had normal body weight, no abnormal performance, and were generally in good condition.

6. Summary

The test drugs GB226 (5mg/kg), Fruquintinib high-dose (2mg/kg), GB226+ Fruquintinib high-dose (2mg/kg), GB226+ Fruquintinib low-dose (1mg/kg) for PD- 1 Humanized MC38 mouse tumor models have significant anti-tumor effects, effectively inhibiting tumor growth, and the anti-tumor effect of the combination of furquintinib and GB226 is significantly better than the corresponding single-agent therapy. This shows that the combination of anti-PD-1 antibody GB226 and fruquintinib has a certain synergistic effect, and compared with the corresponding single drug, the effect of inhibiting tumor growth is obvious. During the administration of GB226 and Fruquintinib, the animals were well tolerated and no adverse reactions were seen.

Example 2

Phase Ib clinical trial of anti-PD-1 antibody combined with Fruquintinib in the treatment of metastatic colorectal cancer

Research purposes

main purpose:

●To evaluate the safety and tolerability of GB226 combined with Fruquintinib in the treatment of metastatic colorectal cancer (mCRC)

●Obtain the recommended dose of GB226 combined with Fruquintinib in the treatment of metastatic colorectal cancer (mCRC) in future clinical studies

Secondary purpose :

●Evaluate the pharmacokinetic (PK) characteristics of GB226 combined with Fruquintinib

●Preliminary evaluation of the anti-tumor activity of GB226 combined with Fruquintinib in the treatment of mCRC

●Evaluate the immunogenicity of GB226

Exploratory purpose :

Explore the expression of programmed cell death protein ligand-1 (PD-L1) in tumor tissues of patients with mCRC, as well as microsatellite instability (MSI) and/or mismatch repair defects (dMMR), tumor mutational burden (TMB), etc. The correlation between biomarkers and the clinical efficacy of GB226 combined with Fruquintinib.

standard constrain:

It is planned to recruit at least 21 patients with metastatic colorectal cancer who have failed first-line treatment or above.

The following standards need to be met:

1. Age from 18 to 75 years old, regardless of gender;

2. Understand the test procedures and content, and voluntarily sign written informed consent;

3. Patients with colorectal cancer diagnosed by histopathology;

4. Patients with metastatic colorectal cancer who have previously received first-line or more treatment failures. Treatment failure refers to disease progression or intolerable toxicity after receiving ≥1 cycle of treatment; or recurrence during adjuvant chemotherapy/neoadjuvant therapy or within 6 months after the end of treatment;

5. ECOG score 0-1;

6. Expected survival ≥ 3 months;

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

8. Before the first study medication, complete systemic chemotherapy, targeted therapy or other anti-tumor biological treatments (tumor vaccines, cytokines or growth factors for the purpose of tumor control) for at least 4 weeks (oral fluorouracil drugs should be stopped for at least 2 weeks ); Complete systemic or local palliative radiotherapy for at least 4 weeks; have not received anti-angiogenesis small molecule targeted drugs in the past;

9. Before the first study medication, the systemic corticosteroid medication (prednisone> 10 mg/day or equivalent dose) has been discontinued for at least 2 weeks;

10. Before the first study medication, major operations requiring general anesthesia must have been completed at least 8 weeks; operations requiring local anesthesia/epidural anesthesia must have been completed at least 4 weeks;

11. Blood routine requires hemoglobin ≥90g/L (blood transfusion is not allowed within 14 days before baseline blood routine examination), and neutrophils ≥1.5×10 ⁹ /L (recombinant human granulocyte colonies are not used within 14 days before baseline blood routine examination Stimulating factor support therapy), platelets ≥100×10 ⁹ /L (recombinant human thrombopoietin or blood transfusion was not used for supportive therapy in the 14 days before the baseline blood routine examination);

12. Serum creatinine≤1.5×ULN or calculated value of creatinine clearance ≥60mL/min (Cockcroft-Gault formula), and urine protein <2+ or <1.0g/L. For patients with urine protein ≥2+ or ≥1.0g/L at baseline, a 24-hour urine protein quantitative test should be performed and must be ≤1.0g/L before they can be selected;

13. Total bilirubin ≤ 1.5 × ULN (unless it is confirmed to have Gilbert syndrome), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 2.5 × ULN (patients with liver metastases allow AST and/or ALT ≤5×ULN);

14. Thyroid function indicators: Thyroid-stimulating hormone (TSH) and free thyroxine (FT3/FT4) are in the normal range; if TSH is not in the normal range and FT3/FT4 is in the normal range, you can join the group;

15. Adverse reactions caused by previous treatments were restored to grade 1 or below before enrollment (except for hair loss and neurotoxicity ≤ grade 2 caused by chemotherapy drugs);

16. Women who are not pregnant within 7 days before administration, and men or women during the reproductive period need to agree to take medically approved effective contraceptive measures during the entire trial period and within 6 months after the end of the trial;

17. Patients can be followed up on schedule, can communicate well with the investigator, and can complete the study in accordance with the research regulations.

Exclusion criteria:

If any one of the following criteria is met, the research 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 included in the group (at least 4 weeks after radiotherapy, no progression and/or no after surgical resection Neurological symptoms or signs do not require treatment with glucocorticoids, antiepileptic drugs, anticonvulsants or mannitol);

2. Patients who have previously suffered from other malignant tumors (except for cured cervical carcinoma in situ and skin basal cell carcinoma) are not allowed to participate in the study unless he/she is in complete remission for at least 2 years before enrollment and does not need other treatment Or no other treatment is required during the study period;

3. A history of active and known autoimmune diseases, including but not limited to systemic lupus erythematosus, psoriasis, rheumatoid arthritis, inflammatory bowel disease, Hashimoto's thyroiditis, etc., except: Type I Diabetes, hypothyroidism that can be controlled only by hormone replacement therapy, skin diseases that do not require systemic treatment (such as vitiligo, psoriasis), and controlled celiac disease;

4. Have used anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody or anti-CTLA-4 antibody treatment (or any other antibody that acts on T cell co-stimulation or checkpoint pathway);

5. Uncontrolled hypertension (systolic blood pressure ≥140mmHg and/or diastolic blood pressure ≥90mmHg) or pulmonary hypertension or unstable angina pectoris; have had myocardial infarction or have undergone coronary artery bypass surgery or coronary artery bypass surgery within 6 months before administration Stent implantation surgery; New York Heart Association (NYHA) standard grade 3-4 history of chronic heart failure; clinically significant valvular disease; severe arrhythmia requiring treatment, including QTc interval ≥450ms for men and ≥470ms for women( Calculated by Fridericia formula); Cerebrovascular accident (CVA) or transient ischemic attack (TIA) within 12 months before administration;

6. Patients with a history of arterial thrombosis or deep vein thrombosis within 6 months before the first study medication, or with evidence of bleeding tendency or medical history within 2 months before the 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 mucosal ulcers or fractures that have not healed completely;

8. The image shows signs of tumor invasion of large blood vessels, including the tumor has completely approached, surrounded or invaded the lumen of large blood vessels (such as pulmonary artery or superior vena cava);

9. Those who have difficulty swallowing or have known drug absorption disorders;

10. Researchers believe that gastrointestinal disorders that will significantly affect the absorption of oral drugs or conditions that may cause gastrointestinal bleeding or perforation (such as duodenal ulcer, intestinal obstruction, acute Crohn’s disease, ulcerative colitis, large-area stomach And small bowel resection, etc.). Patients with chronic Crohn's disease and ulcerative colitis (except for total colon and rectal resection) should be excluded even in the inactive period. People with hereditary non-polyposis colorectal cancer or familial adenomatous polyposis syndrome. Those who have a history of intestinal perforation and intestinal fistula, but have not recovered after surgical treatment;

11. Suffer from interstitial pneumonia now or in the past;

12. Uncontrollable pleural and abdominal cavity and pericardial effusions that require repeated drainage or have obvious symptoms;

13. Active infection 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> the upper limit of normal value of detection unit; hepatitis B surface antigen (HBsAg) positive and HBV-DNA copy Number> upper limit of normal value of detection unit;

15. Comorbidities that need to be treated with immunosuppressive drugs, or comorbidities that require systemic or topical corticosteroids at an immunosuppressive dose (prednisone> 10 mg/day or equivalent dose of similar drugs);

16. It is expected that live vaccines or attenuated vaccines will be given 4 weeks before the start of the study drug use, during the treatment period, or within 5 months of the last administration;

17. Those who have a history of drug abuse or drug abuse upon questioning;

18. Breastfeeding women (agree to stop breastfeeding during the trial period and join the group);

19. Within 30 days before starting to use the study drug or within 5 half-lives of other test drugs (whichever is shorter), received treatment with other experimental drugs or used experimental devices within 30 days;

20. Known to be allergic to the study drug or any of its excipients; Known to have a history of severe allergic diseases;

21. Other situations that the investigator believes are not suitable for participating in this clinical trial.

Test drug

GB226 injection:

The GB226 preparation is a colorless to light yellow liquid, the specification is 70mg/7ml/bottle, GB226 (that is, the anti-PD-1 antibody with heavy and light chain sequences as shown in SEQ ID NO: 9 and SEQ ID NO: 10 of the present invention) Prepared in 100ml of 0.9% sodium chloride solution, the concentration of administration must be controlled at 1mg/ml～10mg/ml, and the infusion is completed within 60 minutes ± 10 minutes after the first application. If there is no infusion reaction, it can be adjusted to ≥30 minutes later.

Fruquintinib capsules: two sizes, 1mg or 5mg, orally on an empty stomach, once a day.

Method of administration

Subjects received GB226 combined with Fruquintinib treatment (see Table 7 for specific dosage and method) until confirmed disease progression, intolerable toxicity, withdrawal of informed consent, start of other anti-tumor treatments, loss to follow-up, death, The investigator or subject decides to terminate the treatment or withdraw from the study, or the study ends.

Table 7. The dosing schedule of GB226 combined with Fruquintinib

^* : If there is intolerable toxicity in the first dose group, the researcher and the sponsor can reduce the dose, such as fruquintinib: 2mg, 1mg/day for exploration;

#: If the second dose group is safe, the investigator and the sponsor can increase the dose after discussion (fruquintinib to 5mg/day)

Primary endpoints and evaluation indicators:

●The incidence and characteristics of adverse events, as well as laboratory examination results, physical examination, 12-lead electrocardiogram, and changes in vital signs compared with baseline

●Dose limiting toxicity (DLT)

●Maximum tolerated dose (MTD) or recommended dose for extended period (RDE)

Secondary endpoints and evaluation indicators:

●The evaluation indicators of clinical effectiveness include objective response rate (ORR), disease control rate (DCR), duration of response (DOR), progression-free survival (PFS), and overall survival (OS)

●Pharmacokinetic (PK) evaluation index. The pharmacokinetic parameters of GB226 include T _max , C _max , C _{ss, min} , R _{C, trough,} etc. The _{pharmacokinetic parameters of Fruquintinib include T max} , C _max , AUC _0-24h , _Rauc etc.

●Immunogenicity of GB226: the number and percentage of subjects who produced anti-GB226 antibodies (ADA) and neutralizing antibodies (NAb)

Exploratory endpoints and evaluation indicators:

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

Clinical outcome

Statistical analysis method

Use descriptive statistics to summarize. In general, continuous variables (such as age) will use the number of observations, mean, median, standard deviation, minimum and maximum values for statistical description; categorical variables will use frequency and their percentages for statistical description. All variables obtained at each observation time point were statistically described by dose group.

All adverse events (including deaths and serious adverse events) will be listed and summarized in accordance with the dose group. MedDRA will be used to code the adverse events according to organ systems and standard terms, and the NCI CTC AE 4.03 version will classify the adverse events. Abnormal list and summary of laboratory examinations, vital signs, electrocardiogram examinations and other safety indicators.

Descriptive statistical methods were used to analyze the efficacy endpoints. The Clopper-pearson method is used to estimate ORR and DCR and provide the corresponding 90% confidence interval. If the number of subjects in the dose group exceeds 10, the Kaplan-Meier method is used to analyze PFS and DOR, and the median and 90% confidence interval are provided.

Pharmacokinetic analysis:

Blood drug concentration (c)-time (t) data analysis: PK concentration set is used to draw individual and average ct curves; the number of cases, mean, standard deviation, median, maximum, and maximum value of blood drug concentration at time points are listed. Minimum value and coefficient of variation, etc. PK parameter analysis: The PK parameter analysis set is used to calculate the pharmacokinetic parameters of each subject from the non-compartmental model, including GB226 T _max , C _max , C _{ss, min} , R _{C, trough,} etc., Fruquintinib T _max , C _max , AUC _0-24h , _Rauc and so on. Calculate the number of cases, arithmetic mean, standard deviation, coefficient of variation, quartile, maximum, minimum and geometric mean of each parameter at the same time.

Effectiveness summary

As of November 29, 2020, the study is in the dose-escalation phase, and a total of 15 subjects were enrolled. As shown in Table 8, 3 subjects were enrolled in the first dose group, 6 subjects were enrolled in the second dose group, and 6 subjects were enrolled in the third dose group. In this study, there were 11 cases of colon cancer and 4 cases of rectal cancer.

Table 8 Study medication status and best curative effect (BOR) of the enrolled subjects

In this study, as of November 29, 2020, a total of 12 subjects had received the combination therapy of GB226 and furoquine and had at least one tumor evaluation. The overall disease control rate (DCR) was 91.7% (61.5- 99.8%), including 3 cases of PR (remission of disease), 8 cases of SD (stable disease), and 1 case of PD (progressive disease). The objective response rate (ORR) was 25% (95% CI 0.0548-0.5719), and mPFS (progression-free survival) was 9.66 months (1.81-16.46+).

The details of 3 subjects who obtained PR are as follows:

1. 01-002: MSI-H/dMMR type, second-line treatment failed, ECOG score 1 point. The subject’s first tumor evaluation was SD, and PR occurred in the second tumor evaluation. After that, the tumor was progressively reduced. So far, 9 tumor evaluations have been done. The tumor has shrunk by 72.4% compared with baseline, and the lesion has been reduced from 85mm to 29mm. PR time It lasts for 16 months and is currently under study.

2. 01-007: MSS/pMMR type, second-line treatment failed, ECOG score 1 point. The subject showed a progressive reduction in the therapeutic effect, but an immune rash appeared after 6 months of study medication. Hormone therapy was stopped for 2 months. During the withdrawal, the tumor was evaluated as PR, and the tumor was reduced from 32mm to 20mm, a reduction of 37.5%. It lasts for 4 months and is currently under study.

3. 02-002: MSS/pMMR type, third-line treatment failed, ECOG score 1 point. Due to the subject's AE with elevated transaminase, Fruquintinib was reduced to 4 mg QD. The first evaluation of the efficacy was PR, the tumor shrank by 46%, lasting 1.7 months, and the drug is still under study.

An analysis of the curative effect of 12 subjects with different types of MSI and/or MMR showed that the combined treatment regimen in this study showed a good curative effect on both MSI-H/dMMR and MSS/pMMR patients. According to the existing research results at home and abroad, the MSS/pMMR type is considered to be a cold tumor, that is, patients of this type have low or no response to most immunotherapy regimens, but there are 2 MSS/pMMR patients in this study All patients responded to treatment and achieved disease remission. The best efficacy of the other 8 MSS/pMMR subjects was SD, and only one MSS/pMMR subject showed PD. One patient with MSI-H/dMMR type had the best curative effect to achieve PR.

Security summary

As of November 29, 2020, 9 subjects in the first and second dose groups have completed 28-day DLT observation. During the DLT observation period, no DLT event was observed. In the third dose group, 2 cases of DLT (grade 3 adverse events) were observed in the third dose group. After the suspension of the administration of GB226 and Fruquintinib in these 2 cases, and the corresponding treatment, the adverse events could be recovered normal.

The main adverse reactions in the subjects in this study were: hypertension, proteinuria, abnormal liver function (increased transaminase and bilirubin), abnormal thyroid function (hypothyroidism, hyperthyroidism, decreased T4, decreased TSH, etc.), hands and feet Syndrome, skin rash. In addition, there are adverse events such as dysphonia, tinnitus, abdominal pain, diarrhea, fatigue, loss of appetite, weight loss, etc., all of which have been identified risks for GB226 and Fruquintinib, and no new safety events and toxicity have occurred. Security incidents.

In summary, the current clinical study of Genostomab Injection (GB226) combined with Fruquintinib in the treatment of metastatic colorectal cancer has obtained preliminary efficacy, and its safety risk is also controllable.

Example 3

Safety of anti-PD-1 antibody combined with furquintinib in the treatment of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) treatment failure in patients with relapsed or metastatic non-small cell lung cancer (NSCLC) with EGFR-sensitive mutations Phase Ib with extended phase clinical trials of sex and effectiveness

The trial population was relapsed or metastatic NSCLC patients with EGFR-sensitive mutations who failed EGFR-TKI treatment.

main purpose:

●Evaluate the safety and tolerability of GB226 combined with Fruquintinib in the treatment of EGFR-TKI-treated patients with recurrent or metastatic NSCLC with EGFR-sensitive mutations, and determine the maximum tolerated dose (MTD) or the recommended extended dose (RDE)

Secondary purpose:

●Evaluate the pharmacokinetic (PK) characteristics of GB226 combined with Fruquintinib

●Preliminary evaluation of the anti-tumor activity of GB226 combined with Fruquintinib in the treatment of recurrent or metastatic NSCLC with EGFR-TKI failure

●Evaluate the immunogenicity of GB226 combined with Fruquintinib in the treatment of recurrent or metastatic NSCLC with EGFR-TKI failure

Purpose of Exploration:

Programmed cell death protein ligand-1 (PD-L1) and microsatellite instability (MSI)/mismatch repair defect (dMMR) in tumor tissues of patients with recurrent or metastatic NSCLC with EGFR-sensitive mutations that have failed EGFR-TKI treatment The correlation between biomarkers such as tumor mutation burden (TMB) and the clinical efficacy of GB226 combined with Fruquintinib.

standard constrain:

Only those who meet all of the following conditions can be selected for this trial.

1. 18 to 75 years old, no gender limit;

2. Understand the test procedures and content, and voluntarily sign a written informed consent form, have good compliance, and cooperate with the follow-up;

3. Recurrent or metastatic NSCLC confirmed by histology or cytology;

4. Confirm that the EGFR gene sensitive mutation is positive and meet one of them: exon 19 deletion (19DEL), exon 21 point mutation (L858R/L861Q), exon 18 point mutation (G719X), 20 exon Sub-point mutation (S768I), and meet the following conditions:

a. After EGFR-TKI treatment fails, there is no T790M mutation detected;

b. Patients with T790M mutation detected after the failure of EGFR-TKI treatment and failed to receive the third-generation EGFR-TKI treatment; patients with primary T790M mutation, who have progressed after receiving the third-generation EGFR-TKI treatment or no other effective treatment drugs;

c. The above patients have failed chemotherapy or are unwilling or intolerant to chemotherapy

5. According to the RECIST 1.1 standard, the subject must have at least one measurable target lesion (lesions with longest diameter ≥ 10 mm, or lymph nodes with short diameter ≥ 15 mm) that can be examined by CT or MRI;

6. Expected survival period ≥ 3 months;

7. ECOG physical status score 0 to 1 points;

8. Systemic chemotherapy, monoclonal antibody drug therapy, radical/extensive radiotherapy, and previous anti-tumor biotherapy (tumor vaccine, cytokine or growth factor for tumor control) have been completed for at least 4 weeks before trial medication; 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. The EGFR-TKI treatment has been completed for more than 2 weeks before the trial drug;

10. Have not received Fruquintinib treatment in the past;

11. Before trial medication, major operations requiring general anesthesia must have been completed for at least 8 weeks; operations requiring local anesthesia/epidural anesthesia must have been completed for at least 4 weeks, and the patient has recovered;

12. Before the trial medication, the corticosteroid medication (prednisone>10mg/day or equivalent dose) used systemically has been discontinued for at least 2 weeks;

13. The laboratory tests performed by the screening must meet the following standards:

●Blood routine (no blood transfusion, no use of G-CSF, no correction with medication within 14 days before screening):

1) Hemoglobin HGB≥90g/L;

2) Absolute neutrophil count ANC≥1.5×10 ⁹ /L;

3) Platelet PLT≥100×10 ⁹ /L;

●Clinical Biochemistry:

1) Total bilirubin (TBIL) ≤ 1.5 times × upper limit of normal [ULN] (Gilbert syndrome allows ≤ 5 times × ULN);

2) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 2.5 times × ULN (patients with liver metastases allow AST and/or ALT ≤ 5 times × ULN);

3) Serum creatinine (Cr)≤1.5 times×ULN or endogenous creatinine clearance ≥50mL/min (Cockcroft-Gault formula);

4) Urine protein <2+ or <1.0g/L. For patients with urine protein ≥2+ or ≥1.0g/L at baseline, a 24-hour urine protein quantitative test should be performed and must be ≤1.0g/L before they can be selected;

●Cagulation function:

1) Activated partial thromboplastin time (APTT) or prothrombin time (PT) ≤ 1.5 times × ULN;

14. Thyroid function indicators: Thyroid-stimulating hormone (TSH) and free thyroxine (FT3/FT4) are in the normal range; if TSH is not in the normal range, FT3/FT4 can be included in the normal range;

15. Adverse reactions caused by previous treatments were restored to grade 1 or below before enrollment (except for hair loss and neurotoxicity ≤ grade 2 caused by chemotherapy drugs);

16. Women who are not pregnant within 7 days before administration, and men or women during the reproductive period need to agree to take medically approved effective contraceptive measures during the entire trial period and within 6 months after the end of the trial;

17. Subjects need to provide tissue samples and are willing to undergo tissue biopsy when needed.

Exclusion criteria:

Those who meet one of the following conditions will not be selected for this test.

1. Patients with lung squamous cell carcinoma (including adenosquamous carcinoma);

2. The genetic test results show that the ALK fusion gene is rearranged;

3. Patients who have previously suffered from other malignant tumors (except for cured cervical carcinoma in situ and skin basal cell carcinoma or squamous cell carcinoma) shall not participate in the trial unless he/she has been cured for at least 5 years before enrollment, and It is estimated that no other anti-tumor treatments will be required during the entire trial period;

4. Imaging (CT or MRI) shows that the tumor focus is less than 5 mm from the large blood vessels, or there is a central type tumor that invades the local large blood vessels; or it shows that there are obvious cavitation or necrotic tumors in the lung;

5. Active central nervous system (CNS) metastasis, including symptomatic brain metastasis or meningeal metastasis or spinal cord compression, etc.; asymptomatic brain metastasis can be included in the group (at least 4 weeks after radiotherapy, no progression and/or no after surgical resection There are neurological symptoms or signs that do not require treatment with glucocorticoids, anticonvulsants or mannitol);

6. Symptomatic and difficult to control serous effusion such as abdominal effusion, pleural effusion or pericardial effusion;

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

8. Currently (within 10 days before the first use of the study drug) is receiving thrombolytic therapy or therapeutic use of anticoagulant drugs (except for preventive use of anticoagulant drugs);

9. A history of active and known autoimmune diseases, including but not limited to systemic lupus erythematosus, psoriasis, rheumatoid arthritis, inflammatory bowel disease, Hashimoto’s thyroiditis, etc., except: Type I Diabetes, hypothyroidism that can be controlled only by hormone replacement therapy, skin diseases that do not require systemic treatment (such as vitiligo, psoriasis), and controlled celiac disease.

10. Have used anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody or anti-CTLA-4 antibody treatment before (or any other antibody that acts on T cell co-stimulation or checkpoint pathway);

11. Uncontrolled hypertension (systolic blood pressure ≥140mmHg and/or diastolic blood pressure ≥90mmHg) or pulmonary hypertension or unstable angina; have had myocardial infarction or have undergone bypass or stent surgery within 6 months before the administration; New York Heart Association (NYHA) standard 3-4 grade chronic heart failure history; severe arrhythmia requiring treatment, including QTc interval ≥450ms for men and ≥470ms for women (calculated by Fridericia formula); 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 mucosal ulcers or fractures that have not healed completely;

13. Difficulty in swallowing or gastrointestinal disorders that the researchers believe will significantly affect the absorption of oral drugs or conditions that may cause gastrointestinal bleeding or perforation (such as duodenal ulcer, gastrointestinal obstruction, clinically active diverticulitis, intra-abdominal abscess , Peritoneal cancer metastasis, acute Crohn’s disease, ulcerative colitis, extensive gastric and small bowel resection, etc.). Patients with chronic Crohn's disease and ulcerative colitis (except for total colon and rectal resection) should be excluded even in the inactive period. People with hereditary non-polyposis colorectal cancer or familial adenomatous polyposis syndrome. Those who have a history of intestinal perforation and intestinal fistula, but have not recovered after surgical treatment;

14. Past or current active tuberculosis infection, or other active infection requiring systemic treatment;

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

16. Comorbidities that need to be treated with immunosuppressive drugs, or comorbidities that need to be treated systemically at an immunosuppressive dose (prednisone> 10 mg/day or equivalent dose of similar drugs), in the absence of active autoimmune diseases Under circumstances, inhaled or topical steroids and doses >10mg/day of prednisone or equivalent doses of similar drugs are allowed;

17. History of interstitial lung disease;

18. Within 30 days before the start of the use of the test drug or within 5 half-lives of other test drugs (whichever is shorter), received treatment with other test drugs or used experimental devices within 30 days;

19. Live vaccines or attenuated vaccines are expected to be given 4 weeks before administration, during treatment, or 5 months after the last administration;

20. Those who have a history of drug abuse or drug abuse upon questioning;

21. Women who are breastfeeding;

22. Known to be allergic to recombinant humanized PD-1 monoclonal antibody or any of its excipients; Known to be allergic to Fruquintinib analogs; Known to have a history of allergic diseases or have severe allergies;

23. The investigator believes that it is not suitable for participating in this clinical trial for various other reasons.

Test drug

GB226 injection:

The GB226 preparation is a colorless to light yellow liquid, the specification is 70mg/7ml/bottle, GB226 (that is, the anti-PD-1 antibody with heavy and light chain sequences as shown in SEQ ID NO: 9 and SEQ ID NO: 10 of the present invention) Prepared in 100ml of 0.9% sodium chloride solution, the concentration of administration must be controlled at 1mg/ml～10mg/ml, and the infusion is completed within 60 minutes ± 10 minutes after the first application. If there is no infusion reaction, it can be adjusted to ≥30 minutes later.

Fruquintinib capsules: two sizes, 1mg or 5mg, orally on an empty stomach, once a day.

Method of administration

Dosage during dose escalation phase:

Primary endpoint:

●The frequency and characteristics of adverse events (AE) and serious adverse events (SAE), as well as changes in laboratory examinations, vital signs, electrocardiogram and physical examination compared with baseline, etc.

●Dose limiting toxicity (DLT)

●Maximum tolerated dose (MTD) or recommended dose for extended period (RDE)

Secondary endpoint:

●GB226 pharmacokinetic (PK) evaluation indicators, including: peak concentration (C _max ), peak time (T _max ), area under the plasma concentration-time curve (AUC _0-t and AUC _0-∞ ) , Apparent volume of distribution (V _d ), mean residence time (MRT), total clearance (CL); and the area under the plasma concentration-time curve (AUC _0-τ ) and average steady-state plasma concentration during the interval between medications (C _avg ), minimum blood concentration (C _min ) and steady-state clearance (CL _ss )

●Furaquintinib blood concentration

●Efficacy evaluation indicators include: objective response rate (ORR), disease control rate (DCR), duration of response (DOR), progression-free survival (PFS) and overall survival (OS)

●Immunogenicity of GB226: the number and percentage of subjects who produced anti-GB226 antibodies (ADA) and neutralizing antibodies (NAb)

Explore the end point:

Programmed cell death protein ligand-1 (PD-L1) protein expression, microsatellite instability (MSI)/mismatch repair defect (dMMR), tumor mutational burden (TMB) and efficacy (ORR, DOR, DCR, PFS, OS) relevance.

Clinical outcome

Statistical analysis method

Use descriptive statistics to summarize. In general, continuous variables (such as age) will use the number of observations, mean, median, standard deviation, minimum and maximum values for statistical description; categorical variables will use frequency and their percentages for statistical description. All variables obtained at each observation time point were statistically described by dose group.

All adverse events (including deaths and serious adverse events) will be listed and summarized in accordance with the dose group. MedDRA will be used to code the adverse events according to organ systems and standard terms, and the NCI CTC AE 4.03 version will classify the adverse events. Abnormal list and summary of laboratory examinations, vital signs, electrocardiogram examinations and other safety indicators.

Descriptive statistical methods were used to analyze the efficacy endpoints. The Clopper-pearson method is used to estimate ORR and DCR and provide the corresponding 90% confidence interval. If the number of subjects in the dose group exceeds 10, the Kaplan-Meier method is used to analyze PFS and DOR, and the median and 90% confidence interval are provided.

Effectiveness summary

As of October 15, 2020, a total of 12 subjects were enrolled in the study. The best efficacy evaluation is shown in Table 9. Among the 12 subjects, 10 subjects received at least one tumor evaluation, and 2 of them were observed The best curative effect was disease remission (PR), the best curative effect observed in 4 cases was stable disease (SD), and the best curative effect observed in 4 cases was disease progression (PD). The overall objective response rate (ORR) is 20%, and the disease control rate (DCR) is 60%.

Table 9 Research medication status and best curative effect of the enrolled subjects

*As of the data cut-off time, the 01-016 subject did not reach the time for the first imaging examination, and the 01-017 subject was dropped out of the study due to loss to follow-up before the first imaging examination

Security summary

As of October 15, 2020, 3 subjects in the first dose group and 3 subjects in the second dose group have completed 28 days of DLT observation, and no DLT event was observed during the DLT observation period. One of the 6 subjects in the third dose group had a DLT event during the DLT observation period, which was grade 3 proteinuria. The study drug was taken to suspend the medication, and after oral treatment with Shenyankang tablets, furosemide, spironolactone, and Huangqi granules get well. No DLT event was observed during the DLT observation period in the remaining 5 subjects.

As of October 15, 2020, common (incidence ≥10%) adverse reactions in subjects mainly include: palmoplantar swelling syndrome, proteinuria, elevated alanine aminotransferase, and aspartate amino transfer Increased enzymes, decreased neutrophil count, and fatigue. In addition, adverse reactions with an incidence of <10% include: decreased white blood cell count, gum pain, hypoproteinemia, abnormal liver function, hyperuricemia, hypertension, increased conjugated bilirubin, cough, skin ulcers , Peripheral edema, pleural effusion, increased blood bilirubin, decreased platelet count, sinus tachycardia, epistaxis, hyperglycemia, hypothyroidism, hyperthyroidism, decreased thyroxine, blood in the urine, palpitations , Elevated blood thyroid stimulating hormone, skin rash, elevated blood alkaline phosphatase adverse reactions. Current research data shows that GB226 (210mg) combined with Fruquintinib (2mg, 4mg, or 5mg) has acceptable safety in subjects with EGFR-sensitive mutations that have failed EGFR-TKI treatment with recurrent or metastatic non-small cell lung cancer. Tolerability and known adverse reactions are risks that have been identified by GB226 and Fruquintinib, and there have been no new safety events and safety events caused by superimposed toxicity.

In summary, GB226 combined with Fruquintinib in the treatment of EGFR-TKI failed EGFR-TKI treatment of EGFR-sensitive mutation recurrence or metastatic non-small cell lung cancer clinical research safety risk is controllable, and has obtained preliminary efficacy, the study will select the third dose group As the recommended dose for the expansion phase, the subsequent dose expansion phase is carried out.

The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited to this. Anyone familiar with the technical field within the technical scope disclosed by the present invention, according to the technical solution of the present invention Equivalent replacements or changes to the improved conception thereof shall all fall within the protection scope of the present invention.

Claims (27)

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

   The 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,

   The 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,

   The light chain CDR3 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: 3,

   The 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,

   The 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

   The 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 according to 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 1, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a light chain variable region sequence, which is an amino acid sequence as shown in SEQ ID NO: 7, or with an amino acid sequence as shown in SEQ ID The amino acid sequence shown by NO: 7 has at least 80% homology.
4. The use according to claim 1, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region sequence, which is an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence as shown in SEQ ID The amino acid sequence shown by NO: 8 has at least 80% homology.
5. The use according to claim 1, wherein the heavy chain sequence of the anti-PD-1 antibody is the sequence shown in SEQ ID NO: 9 and the light chain sequence is the sequence shown in SEQ ID NO: 10.
6. The use according to claim 1, wherein the cancer has one or more of the following characteristics:

   Express PD-L1, high degree of microsatellite instability/mismatch repair defect and high tumor mutation burden.
7. The use according to claims 1-6, wherein the cancer is lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, bowel 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, pancreatic cancer, prostate cancer, kidney cancer or a combination thereof.
8. The use according to claim 7, wherein the bowel cancer is colorectal cancer.
9. The use according to claim 8, wherein the colorectal cancer is metastatic colorectal cancer.
10. The use according to claim 9, wherein the metastatic colorectal cancer is microsatellite stable and/or complete mismatch repair function or high degree of microsatellite instability and/or mismatch repair defect.
11. The use according to claim 7, wherein the lung cancer is non-small cell lung cancer.
12. The use according to claim 11, wherein the non-small cell lung cancer is recurrent or metastatic non-small cell lung cancer with a sensitive mutation of EGFR.
13. The use according to claim 12, wherein the recurrent or metastatic non-small cell lung cancer with EGFR sensitive mutations has failed EGFR-TKI treatment.
14. The use according to claim 13, wherein the recurrent or metastatic non-small cell lung cancer with EGFR sensitive mutation has one or more of the following characteristics:

    Exon 19 deletion (19DEL), exon 21 point mutation (L858R/L861Q), exon 18 point mutation (G719X) and exon 20 point mutation (S768I).
15. The use according to any one of claims 1-14, wherein the single administration dose of the anti-PD-1 antibody or antigen-binding fragment thereof is 0.1-40 mg/kg individual body weight or a single fixed dose of 100-400 mg.
16. The use according to claim 15, wherein the single administration dose of the anti-PD-1 antibody or antigen-binding fragment thereof is 3 mg/kg body weight or a single fixed dose of 210 mg.
17. The use according to claim 15 or 16, wherein the frequency of administration of the anti-PD-1 antibody or antigen-binding fragment thereof is once every two weeks.
18. The use according to claim 15, wherein the single administration dose of the anti-PD-1 antibody or antigen-binding fragment thereof is 5 mg/kg individual body weight.
19. The use according to claim 18, wherein the frequency of administration of the anti-PD-1 antibody or antigen-binding fragment thereof is twice a week.
20. The use according to any one of claims 1-19, wherein the single administration dose of Fruquintinib is 0.1-10 mg/kg individual body weight or a single fixed dose of 0.1-20 mg.
21. The use according to claim 20, wherein the single administration dose of Fruquintinib is 1 mg/kg or 2 mg/kg of individual body weight, or a single fixed dose of 1 mg, 2 mg, 3 mg, 4 mg or 5 mg is administered.
22. The use according to claim 20 or 21, wherein the frequency of administration of fruquintinib is once a day.
23. The use according to claim 22, wherein the frequency of administration of Fruquintinib is three weeks of continuous administration and one week of drug withdrawal.
24. The use according to any one of claims 8-10, wherein the single administration dose of the anti-PD-1 antibody or antigen-binding fragment thereof is 3 mg/kg individual body weight, and the administration frequency is once every two weeks; And the single fixed dose of Fruquintinib is 3 mg or 4 mg, the frequency of administration is once a day for three consecutive weeks, and the drug is stopped for one week.
25. The use according to any one of claims 11-14, wherein a single fixed dose of the anti-PD-1 antibody or antigen-binding fragment thereof is 210 mg, and the frequency of administration is once every two weeks; and The single fixed dose of Fruquintinib is 5 mg, the frequency of administration is once a day for three consecutive weeks, and the drug is stopped for one week.
26. The use according to any one of claims 1-25, wherein the anti-PD-1 antibody or antigen-binding fragment thereof and fruquintinib or a pharmaceutically acceptable salt thereof are administered in the same administration cycle.
27. A kit containing the anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and fruquintinib or a pharmaceutically acceptable salt thereof.

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